Method for producing purine nucleoside by fermentation

ABSTRACT

A microorganism which has a gene encoding an enzyme in which feedback inhibition is desensitized by substitution of one or two amino acids in PRPP amidotransferase encoded by purF of  Escherichia coli , a gene encoding a protein which is an inactivated repressor of purine nucleotide biosynthesis encoded by purR, a gene encoding an enzyme which is inactivated purine nucleoside phosphorylase encoded by deoD, a gene encoding an enzyme which is inactivated succinyl-AMP synthase encoded by purA, a gene encoding an enzyme which is inactivated 6-phosphogluconate dehydrase encoded by edd, a gene encoding an enzyme which is inactivated phosphoglucose isomerase encoded by pgi and like is bred and a purine nucleoside is produced by culturing the microorganism.

TECHNICAL FIELD

The present invention relates to a method for producing purinenucleosides such as inosine and guanosine which are important as rawmaterials for syntheses of 5′-inosinic acid and 5-guanylic acid,respectively, and a novel microorganism used for the production.

BACKGROUND ART

For the production of inosine and guanosine by fermentation, there havebeen known methods utilizing adenine auxotrophic strains or such strainsfurther imparted with drug resistance against various drugs such aspurine analogues, which strains belong to the genus Bacillus (JapanesePatent Publication Nos. 38-23039 (1963), 54-17033 (1979), 55-2956(1980), and 55-45199 (1980), Japanese Patent Application Laid-Open No.56-162998 (1981), Japanese Patent Publication Nos. 57-14160 (1982) and57-41915 (1982), and Japanese Patent Application Laid-Open No. 59-42895(1984)), or the genus Brevibacterium (Japanese Patent Publication Nos.51-5075 (1976) and 58-17592 (1972), and Agric. Biol. Chem., 42, 399(1978)) and the like.

Conventional acquisition of such mutant strains comprises subjectingmicroorganisms to a mutagenesis treatment such as UV irradiation andnitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine) treatment andselecting a desired strain by using a suitable selection medium. On theother hand, breeding of such mutant strains by the use of geneticengineering techniques have also been practiced for strains belonging tothe genus Bacillus (Japanese Patent Application Laid-Open Nos. 58-158197(1983), 58-175493 (1983), 59-28470 (1984), 60-156388 (1985), 1-27477(1989), 1-174385 (1989), 3-58787 (1991), 3-164185 (1991), 5-84067(1993), and 5-192164 (1993)) and the genus Brevibacterium (JapanesePatent Application Laid-Open No. 63-248394 (1988)).

DISCLOSURE OF INVENTION

The object of the present invention is to create a microorganismsuitable for the production of the purine nucleoside by fermentation.

To achieve the aforementioned object, the present inventors conceived anidea of imparting purine nucleoside-producing ability to a bacterialstrain of the genus Escherichia, which is different in the genus frommicroorganisms which have hitherto been used for the production of thepurine nucleoside by fermentation, and successfully realized it. Thus,the present invention has been completed.

Thus, the present invention provides a microorganism belonging to thegenus Escherichia and having purine nucleoside-producing ability.

Specifically, the present invention provides the microorganism which hasacquired the purine nucleoside-producing ability because of an increaseof an activity of an enzyme involved in the purine nucleosidebiosynthesis in cells of the microorganism. More specifically, thepresent invention provides the microorganism which has acquired thepurine nucleoside-producing ability because of an increase of anexpression amount of a gene for an enzyme involved in the purinenucleoside biosynthesis, and the microorganism which has acquired thepurine nucleoside-producing ability because of deregulation of controlof an enzyme involved in the purine nucleoside biosynthesis.

The enzyme involved in the purine nucleoside biosynthesis may be, forexample, phosphoribosyl pyrophosphate (PRPP) amidotransferase andphosphoribosyl pyrophosphate (PRPP) synthetase.

As a means of desensitizing the control of an enzyme involved in purinenucleoside biosynthesis, for example, deficiency of a purine repressorcan be mentioned.

The present invention further provides the microorganism which hasacquired the purine nucleoside-producing ability because of blockage ofa reaction branching from the purine nucleoside biosynthesis and leadingto another metabolite.

Examples of the reaction branching from the purine nucleosidebiosynthesis and leading to another metabolite include, for example,those catalyzed by an enzyme selected from succinyl-adenosinemonophosphate (AMP) synthase, purine nucleoside phosphorylase, adenosinedeaminase, inosine-guanosine kinase, guanosine monophosphate (GMP)reductase, 5-phosphogluconate dehydrase, phosphoglucose isomerase,adenine deaminase, and xanthosine phosphorylase.

The present invention further provides the microorganism which isenhanced in the purine nucleoside-producing ability because of weakeningof incorporation of a purine nucleoside into cells of the microorganism.

The incorporation of the purine nucleoside into cells of themicroorganism may be weakened by blockage of a reaction involved in theincorporation of the purine nucleoside into cells of the microorganism.An example of the reaction involved in the incorporation of the purinenucleoside into cells of the microorganism is a reaction catalyzed bynucleoside permease.

The present invention further provides a method for producing a purinenucleoside by fermentation comprising culturing the aforementionedmicroorganism in a culture medium to produce and accumulate the purinenucleoside in the medium, and collecting the purine nucleoside.

The present invention described in details below.

(1) Microorganism Belonging to the Genus Escherichia and Having PurineNucleoside-Producing Ability

As examples of the microorganism belonging to the genus Escherichia usedin the present invention, Escherichia coli (E. coli) and the like can bementioned. When E. coli strains are bred by genetic engineeringtechniques, E. coli K12 strain may be utilized.

The term “purine nucleoside” herein used include, for example, inosine,guanosine, and adenosine.

The term “purine nucleoside-producing ability” herein used means abilityto produce and accumulate a purine nucleoside in a medium. The term“having purine nucleoside-producing ability” means that themicroorganism belonging to the genus Escherichia produces andaccumulates a purine nucleoside in a medium in a larger amount than awild strain of E. coli such as W3110 strain, preferably means that themicroorganism produces and accumulates inosine in a medium in an amountof not less than 50 mg/L, more preferably not less than 100 mg/L, stillmore preferably not less than 200 mg/L, most preferably not less than500 mg/L under the condition described in Example 1 below.

In order to breed a microorganism belonging to the genus Escherichia andhaving purine nucleoside-producing ability, it may be adopted breedingby increasing an activity of an enzyme involved in the purine nucleosidebiosynthesis in cells of the microorganism, for example, breeding byincreasing an expression amount of a gene for the enzyme involved in thepurine nucleoside biosynthesis. Alternatively, breeding by desensitizingcontrol of an enzyme involved in the purine nucleoside biosynthesis maybe adopted.

Furthermore, breeding by blocking a reaction branching from purinenucleoside biosynthesis and leading to another metabolite and breedingby weakening of incorporation of a purine nucleoside into cells of themicroorganism.

(2) Microorganism in which an Activity of an Enzyme Involved in PurineNucleoside Biosynthesis in Cells of the Microorganism is Increased

All the enzymes involved in the purine nucleoside biosynthesis and allthe reactions catalyzed by those enzymes in microorganisms belonging tothe genus Escherichia have already been elucidated (Escherichia coli andSalmonella, CELLULAR AND MOLECULAR BIOLOGY, Second Edition vol. 1 andvol. 2, ASM PRESS, WASHINGTON D.C.). The purine nucleoside-producingability can be imparted by increasing an activity of an enzymecatalyzing a rate-limiting reaction among the enzymes. An example of theenzyme catalyzing a reaction of rate-limiting step is PRPPamidotransferase or PRPP synthetase.

Examples of means of increasing an activity of an enzyme involved in thepurine nucleoside biosynthesis in cells are explained below but are notlimited thereto.

As a means of increasing the activity of the enzyme involved in thepurine nucleoside biosynthesis in cells, increasing an expression amountof the gene for the enzyme may be mentioned.

Examples of means of increasing the expression amount of the geneinclude improvement of a regulatory region of the gene and increase of acopy number of the gene, but are not limited thereto.

The improvement of the regulatory region means making modificationthereto to increase a transcription amount of a gene. For example, apromoter can be enhanced by, for example, introducing a mutation intothe promoter to increase a transcription amount of a gene locateddownstream of the promoter. Besides introducing a mutation into apromoter, another promoter which functions in microorganisms such aslac, trp, tac, trc, and PL may be newly introduced. Further, an enhancermay be newly introduced to increase the transcription amount of thegene. Introduction of a gene such as a promoter into chromosome DNA isdescribed in, for example, Japanese Patent Application Laid-Open No.1-215280 (1989).

Specifically, the copy number of the gene may be increased by ligating agene to a multi-copy vector to form a recombinant DNA, and allowing amicroorganism to have the recombinant DNA. The vector includes widelyused ones such as plasmids and phages, and, in addition to these,transposon (Berg, D. E. and Berg, C. M., Bio/Technol., 1, 417 (1983))and Mu phage (Japanese Patent Application Laid-open No. 2-109985(1990)). It is also possible to integrate a gene into a chromosome by amethod utilizing a plasmid for homologous recombination or the like toincrease the copy number of the gene.

For breeding a microorganism belonging to the genus Escherichia andhaving an increased expression amount of a gene for an enzyme involvedin the purine nucleoside biosynthesis, necessary regions of genes may beobtained by amplified by PCR (polymerase chain reaction) mainly based onalready available information about E. coli genes, and used for breedingof the microorganism.

For example, purF, which is a gene coding for PRPP amidotransferase, canbe cloned from the chromosome DNA of E. coli K12 W3110 strain(ATCC27325) using a PCR technique. The chromosome DNA used for this maybe derived from any strain of E. coli. The purF means a gene coding forPRPP amidotransferase, which is subjected to feedback inhibition byadenosine monophosphate (AMP) or guanosine monophosphate (GMP), andincludes mutants generated due to genetic polymorphism and the like.Genetic polymorphism means a phenomenon that an amino acid sequences ofa protein is partially altered due to naturally occurring mutation onthe gene.

As a means of increasing the activity of the enzyme involved in thepurine nucleoside biosynthesis in the cells, it is also possible tointroduce a mutation into a structural gene of the enzyme to enhance theenzymatic activity of the enzyme itself.

As a means of increasing the activity of the enzyme involved in thepurine nucleoside biosynthesis in the cells, it is also possible todesensitize control of the enzyme involved in the purine nucleosidebiosynthesis.

The control of the enzyme involved in the purine nucleoside biosynthesismeans a mechanism negatively controlling the activity of the enzyme, andincludes feedback inhibition by an intermediate in the biosynthesispathway or a final product, attenuation, transcriptional suppression andthe like. A purine nucleoside produced by a microorganism inhibits theactivity of the enzyme involved in the purine nucleoside biosynthesis orrepresses expression of a gene encoding the enzyme through the control.Therefore, for allowing the microorganism to produce the purinenucleoside, it is preferable to desensitize the control.

The enzyme involved in the purine nucleoside biosynthesis, whichundergoes the control, includes PRPP amidotransferase which is subjectedto feedback inhibition by AMP or GMP and PRPP synthetase which issubjected to feedback inhibition by adenosine diphosphate (ADP).Besides, inosine monophosphate dehydrogenase (guaB) and GMP synthetase(guaA) are subjected to feedback inhibition by GMP. Also, a purineoperon, guaBA is subjected to repression.

As a method for desensitizing the control, a method for introducingmutation into a gene encoding the enzyme or a regulatory region thereofmay be mentioned. The mutation includes mutation desensitizing feedbackinhibition, which is usually mutation in a structural gene. The mutationalso includes mutation desensitizing attenuation, which is usuallymutation in attenuator. The mutation also includes mutationdesensitizing repression, which is usually mutation in a gene coding aregulatory protein which is called repressor, or mutation in an operatorregion.

The mutation desensitizing repression includes mutation inactivating apurine repressor. The purine repressor binds to an operator region of apurine operon under the condition that a purine nucleoside exists in alarge amount, resulting in repression of transcription of the operon.Inactivation of the repressor leads to desensitization of therepression.

In order to introduce a mutation into a gene, the site-specificmutagenesis (Kramer, W. and Frits, H. J., Methods in Enzymology, 154,350 (1987)), the recombinant PCR technique (PCR Technology, StocktonPress (1989)), chemical synthesis of a specific portion of DNA,hydroxylamine treatment of a gene of interest, treatment of microbialstrains having a gene of interest by UV irradiation or a chemical agentsuch as nitrosoguanidine or nitrous acid and the like can be used. Whenfunction of a gene should be completely inactivated, addition ordeletion of DNA may be introduced at a suitable restriction site.

A microorganism in which control of an enzyme involved in the purinenucleoside biosynthesis is desensitized can be selected by determiningan expression amount of the enzyme through an enzymatic activity assay,or using antibodies. As an example of a method for obtaining a mutantstrain in which control of an enzyme is desensitized, a methodcomprising selecting a strain growing in a minimal medium containing apurine analogue such as 8-azaadenine and 8-azaguanine, and determiningchange of an expression amount or activity of the enzyme.

(3) Microorganism which has Acquired the Purine Nucleoside-ProducingAbility Because of Blockage of a Reaction Branching from PurineNucleoside Biosynthesis and Leading to Another Metabolite

The purine nucleoside biosynthesis pathway of microorganisms belongingto the genus Escherichia has been already elucidated, and all theenzymes involved in the purine nucleoside biosynthesis and reactionscatalyzed by those enzymes have also been elucidated (Escherichia coliand Salmonella, CELLULAR AND MOLECULAR BIOLOGY, Second Edition, vol. 1and vol. 2, ASM PRESS WASHINGTON D.C.). In addition, some of reactionswhich lead to other metabolites have also been made clear.

A microorganism in which a reaction leading to another metabolite areblocked may become to require the metabolite. In order to cultivate suchmicroorganism that has become to require the metabolite, it is necessaryto add the metabolite or an intermediate (precursor) therefor to aculture medium as a nutrient. Therefore, it is desirable that a reactionnot requiring extra addition of the metabolites when it is blockedshould be selected as the reaction to be blocked.

The purine nucleoside-producing ability may not be always improved byblocking any of the reactions leading to other metabolites. If areaction converting a purine nucleoside intermediate or a purinenucleoside into another metabolite proceeds during the production of thepurine nucleoside by the microorganism, blocking such a reaction mayimprove the purine nucleoside productivity.

A reaction whose blocking may actually improve the purinenucleoside-producing ability may be predicted among the reactionsbranching from the purine nucleoside biosynthesis and leading to theproduction of another metabolite based on the already elucidated schemesof the purine nucleoside biosynthesis.

As a method for blocking the reaction branching from the purinenucleoside biosynthesis and leading to another metabolite, a method fordeleting or inactivating an enzyme catalyzing the reaction or the likemay be mentioned. The enzyme may be deleted, for example, by deleting agene encoding the enzyme. The enzyme may be inactivated by, for example,introducing a mutation into a gene encoding the enzyme, adding an agentspecifically inactivating the enzyme or the like.

Examples of the reaction branching from the purine nucleosidebiosynthesis and leading to another metabolite, whose blocking mayactually improve the purine nucleoside-producing ability, include areaction catalyzed by an enzyme selected from succinyl-AMP synthase,purine nucleoside phosphorylase, adenosine deaminase, inosine-guanosinekinase, GMP reductase, 6-phosphogluconate dehydrase, phosphoglucoseisomerase, adenine deaminase, and xanthosine phosphorylase.

For example, when the branching from IMP to succinyl-AMP and theconversion from inosine to hypoxanthine are blocked, IMP is notconverted to AMP and inosine is not converted to hypoxanthine.Accordingly, it is expected that inosine is accumulated. In order toevaluate the effectiveness of such blocking, a mutant obtained dependingon the purpose may be cultured, and its inosine productivity may bedetermined.

As described in the Examples hereinafter, when E. coli was made adenineauxotrophic by destroying succinyl-AMP synthase gene (purA gene), itbecame necessary to add an AMP substance such as adenine and adenosineto a culture medium for the growth of the adenine auxotroph of E. coli.However, it was found in E. coli that such an added substance wasimmediately converted to inosine or hypoxanthine, and its growth wasceased at a certain point due to the loss of the AMP substance.Therefore, judging from the metabolic pathway of E. Coli, it is expectedthat it is necessary to inactivate adenosine deaminase involved in theconversion of adenosine to inosine or adenine deaminase involved in theconversion of adenine to hypoxanthine as a means of maintaining itsgrowth. Thus, the effectiveness of the inactivation of adenosinedeaminase or adenine deaminase was confirmed, and accumulation ofinosine was observed.

GMP reductase is involved in the conversion of GMP to IMP. It isexpected that guanosine productivity is improved by inactivating the GMPreductase. As shown in Examples below, a certain level of improvement inguanosine accumulation was observed.

A carbon source such as glucose is used for the production of a purinenucleoside. It is known that there is a difference in sugar metabolicsystem leading to purine nucleoside biosynthesis depending on the usedcarbon source or culture conditions. Therefore, for leading themetabolic system to purine nucleoside biosynthesis advantageously, it isconsidered to block branches other than pentose phosphate pathway togive preference in the pentose phosphate pathway. As a means thereof,inactivation of 6-phosphogluconate dehydrase or phosphoglucose isomerasewas tested and the effectiveness thereof was confirmed.

(4) Microorganism which has Acquired the Purine Nucleoside-ProducingAbility by Weakening of Incorporation of a Purine Nucleoside into Cellsof the Microorganism

Since incorporation of a purine nucleoside which has released externallyfrom cells into the cells again is considered unreasonable in view ofenergy for accumulating the purine nucleoside, weakening ofincorporation of a purine nucleoside is effective.

As means of weakening incorporation of a purine nucleoside into cells,blocking of a reaction involved in membrane permeability of the purinenucleoside may be mentioned. The blocking of the reaction can be carriedout in the same manner as described about (3) above.

For example, by inactivating nucleoside permease which is one ofpermeases involved in incorporation of purine nucleosides into cells,improvement in accumulation of inosine was observed.

(5) Method for Producing a Purine Nucleoside

The method for producing a purine nucleoside by fermentation using themicroorganism having the purine nucleoside-producing ability isexplained hereinafter.

Culture medium for purine nucleoside production to be used may be ausual medium containing a carbon source, a nitrogen source, inorganicions and other organic components as required. As the carbon source,saccharides such as glucose, lactose, galactose, fructose, arabinose,maltose, xylose, trehalose, ribose and hydrolysates of starches;alcohols such as glycerol, mannitol and sorbitol; organic acids such asgluconic acid, fumaric acid, citric acid and succinic acid and the likecan be used. As the nitrogen source, inorganic ammonium salts such asammonium sulfate, ammonium chloride, and ammonium phosphate; organicnitrogen such as of soy bean hydrolysates; ammonia gas; aqueous ammoniaand the like can be used. It is desirable that vitamins such as vitaminBI, required substances, for example, nucleic acids such as adenine andRNA, or yeast extract and the like are contained in appropriate amountsas trace amount organic nutrients. Other than these, small amounts ofcalcium phosphate, magnesium sulfate, iron ions, manganese ions and thelike may be added, if necessary.

Cultivation is preferably performed under an aerobic condition for 16 to72 hours, and culture temperature during the cultivation is controlledwithin 30 to 45° C. and pH within 5 to 8. The pH can be adjusted byusing an inorganic or organic acidic or alkaline substance as well asammonia gas.

A purine nucleoside can be recovered from the fermentation liquor by anyor any combination of conventional methods such as techniques utilizingion exchange resin and precipitation.

(6) Specific Examples of Purine Nucleoside-Producing Bacteria

First, purF (a gene coding for PRPP amidotransferase), purR (a genecoding for a purine repressor), deoD (a gene coding for purinenucleoside phosphorylase), purA (a gene coding for succinyl-AMPsynthase), add (a gene coding for adenosine deaminase), gsk (a genecoding for inosine-guanosine kinase), guaC (a gene coding for GMPreductase), edd (a gene coding for 6-phosphogluconate dehydrase), pgi (agene coding for phosphoglucose isomerase), yicP (a gene for coding foradenine deaminase), prs (a gene coding for PRPP synthetase), xapA (agene coding for xanthosine phosphorylase), and nupG (a gene coding fornucleoside permease) are cloned from a chromosome DNA of Escherichiacoli (E. coli) K12 strain W3110 (ATCC27325) by using a PCR technique,and they may be mutated depending on their purposes. The chromosome DNAused for this procedure may be obtained from any strain of E. coli.

The mutation introduced into purF is a mutation for destroying purF or amutation for desensitizing the feedback inhibition of PRPPamidotransferase. The mutation introduced into purR is a mutation fordestroying purR. The mutation introduced into deoD is a mutation fordestroying deoD. The mutation introduced into purA is a mutation fordestroying purA. The mutation introduced into add is a mutation fordestroying add. The mutation introduced into gsk is a mutation fordestroying gsk.

The mutation introduced into guaC is a mutation for destroying guaC. Themutation introduced into edd is a mutation for destroying edd. Themutation introduced into pgi is a mutation for destroying pgi. Themutation introduced into yicP is a mutation for destroying yicP. Themutation introduced into prs is a mutation for desensitizing thefeedback inhibition of PRPP synthetase. The mutation introduced intoxapA is a mutation for destroying xapA. The mutation introduced intonupG is a mutation for destroying nupG.

To introduce a mutation into a gene, the site-specific mutagenesis(Kramer, W. and Frits, H. J., Methods in Enzymology, 154, 350 (1987)),the recombinant PCR technique (PCR Technology, Stockton Press (1989)),chemical synthesis of a specific portion of DNA, hydroxylamine treatmentof a gene of interest, treatment of microbial strains having a gene ofinterest by UV irradiation or a chemical agent such as nitrosoguanidineor nitrous acid and the like can be used. When function of a gene shouldbe completely inactivated, addition or deletion of DNA may be introducedat a suitable restriction site.

Then, purF and prs to which a mutation for desensitizing the feedbackinhibition of PRPP amidotransferase and PRPP synthetase are added,respectively, are introduced as a recombinant DNA into a suitablemicroorganism to express the genes, thereby obtaining a microorganismcontaining the PRPP amidotransferase gene (purF) and the PRPP synthetasegene (prs) whose feedback inhibition is substantially desensitized. Therecombinant DNA obtained above means a vector such as plasmid and phage,into which a useful gene such as the PRPP amidotransferase gene (purF)and the PRPP synthetase (prs) whose feedback inhibition is substantiallydesensitized is integrated as a passenger. The vector may contain apromoter operable in the microorganism, such as lac, trp, tac, trc, andPL so that efficient expression of the useful gene can be obtained.

The recombinant DNA herein used includes any of those obtained byintegrating a useful gene into a chromosome by using a transposon (Berg,D. E. and Berg, C. M., Bio/Technol., 1, 417(1983)), Mu phage (JapanesePatent Application Laid-Open No. 2-109985 (1990)), a plasmid forhomologous recombination or the like.

As the plasmid for homologous recombination, a plasmid having atemperature-sensitive replication origin may be used. The plasmid havingthe temperature-sensitive replication origin can replicate at apermissive temperature, for example, around 30° C., but cannot replicateat a non-permissive temperature, for example, 37° C. to 42° C. In thehomologous recombination method using the plasmid having thetemperature-sensitive replication origin, the plasmid can be replicatedat a permissive temperature, or dropped out at a non-permissivetemperature as required. In the Examples described below, pMAN997, whichcorresponds to pMAN031 (J. Bacteriol., 162, 1196 (1985)) whoseVspI-HindIII fragment is replaced with that of pUC19 (Takara Shuzo)(FIG. 1), was used as the plasmid for homologous recombination.

A specific genetic function on the chromosome was inactivated by thehomologous recombination (Experiments in Molecular Genetics, Cold SpringHabor Lab. (1972)) to improve the purine nucleoside-producing ability.The gene to be inactivated is a gene of which inactivation leadsincrease of an expression amount of a gene for an enzyme involved in thepurine nucleoside biosynthesis. Specifically, the purine repressor gene(purR) on the chromosome was destroyed to remove the expressionregulation mechanism of the purine nucleotide biosynthesis genesincluding the PRPP amidotransferase gene (purF).

Further, a gene coding for an enzyme which catalyzes a reactionbranching from purine nucleoside biosynthesis and leading to anothermetabolite was destroyed. Specifically, the purine nucleosidephosphorylase gene (deoD) was destroyed to suppress the decomposition ofinosine and guanosine to hypoxanthine and guanine, respectively.Furthermore, the succinyl-AMP synthase gene (purA) was destroyed toimpart adenine auxotrophy. Moreover, the adenosine deaminase gene (add)was destroyed to suppress the conversion of adenosine to inosine.Finally, the inosine-guanosine kinase gene (gsk) was destroyed tosuppress the conversion of inosine and guanosine to IMP and GMP,respectively. The GMP reductase gene (guaC) was destroyed to suppressthe conversion of GMP to IMP. The 6-phosphogluconate dehydrase gene(edd) was destroyed to suppress metabolism of sugars through theEntner-Doudoroff pathway. The phosphoglucose isomerase gene (pgi) wasdestroyed to suppress metabolism of sugars through glycolysis pathway,thereby promoting the flow into the pentose phosphate pathway. Theadenine deaminase gene (yicP) was destroyed to suppress the conversionof adenine to hypoxanthine. The xantosine phosphorylase gene (xapA) wasdestroyed to suppress the decomposition of xanthosine to xanthine and tosuppress the decomposition of inosine and guanosine to hypoxanthine andguanine, respectively. The inactivation of a target gene may also beperformed of course by treatment of microbial strains having the geneswith UV irradiation or with a chemical agent such as nitrosoguanidineand nitrous acid.

As the microorganism having the recombinant DNA, a microorganismbelonging to the genus Escherichia in which a gene coding for a targetenzyme such as PRPP amidotransferase was expressed was used.

In order to efficiently utilize the PRPP amidotransferase gene (purF),it is preferably used together with other useful genes, for example,genes involved in the IMP biosynthesis from PRPP other than purF (purD,purT, purL, purM, purK, purE, purC, purB, purH), IMP dehydrogenase gene(guaB), GMP synthetase gene (guaA), PRPP synthetase gene (prs) and thelike. Like the PRPP amidotransferase gene (purF), those useful genes maybe present on a host chromosome, or a plasmid or phage.

A microorganism having deficiency of purA (succinyl-AMP synthase gene)and/or deficiency of deoD (purine nucleoside phosphorylase gene) and/ordeficiency of purR (purine repressor gene) and/or desensitized type purF(PRPP amidotransferase gene) and/or deficiency of add (adenosinedeaminase gene) and/or deficiency of gsk (inosine-guanosine kinase gene)and/or deficiency of guaC (GMP reductase gene) and/or deficiency of edd(6-phosphogluconate dehydrase gene) and/or deficiency of pgi(phosphoglucose isomerase gene) and/or deficiency of yicP (adeninedeaminase gene) and/or deficiency of xapA (xanthosine phosphorylasegene) and/or deficiency of nupG (nucleoside permease gene), or amicroorganism transformed with a recombinant DNA having desensitizedtype PRPP amidotransferase gene (purF) and/or desensitized type prs(PRPP synthetase gene) obtained as described above is cultured so thatthe target purine nucleoside such as inosine and guanosine isaccumulated in the culture medium, and the accumulated nucleoside iscollected.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a construction of pMAN997.

FIG. 2 shows structures of genes for homologous recombination. Numeralsin the figure represent lengths (bp) of obtained fragments and positionsfrom 5′ ends.

FIG. 3 shows structures of genes for homologous recombination. Numeralsin the figure represent lengths (bp) of obtained fragments and positionsfrom 51 ends.

BEST MODE FOR CARRYING OUT THE INVENTION Example 1 1) Breeding of StrainDeficient in PRPP Amidotransferase Gene (purF)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, Perkin Elmer) by usingchromosome DNA of E. coli K12 strain W3110 (ATCC27325) as a template and29-mer and 31-mer primers for both ends, having nucleotide sequences ofCTCCTGCAGAACGAGGAAAAAGACGTATG (SEQ ID NO: 1) andCTCAAGCTTTCATCCTTCGTTATGCATTTCG (SEQ ID NO: 2), and prepared based onthe information of a gene data bank (GenBank Accession No. M26893), andan amplified fragment of about 1530 bp of the purF structural generegion covering SD-ATG and the translation termination codon was clonedinto pCRTMII vector (Invitrogen). The amplified fragment of the PCRproduct can be cloned into this vector as it is. The vector has EcoRIsites as restriction sites at vicinities of the both sides of thecloning site. A PstI site and a HindIII site are respectively providedin the PCR primers.

The cloned 1530 bp purF fragment contained one BglII site at about 880bp from the 5′ end, and pCRTMII vector itself also had one EglII site.Therefore, the plasmid was partially digested with BglII, blunt-ended byT4 DNA polymerase, and then ligated by T4 DNA ligase. Competent cells ofE. coli HB101 were transformed with this ligation solution, andtransformants grown on LB (1% tryptone, 0.5% yeast extract, 0.1% NaCl,0.1% Glucose, pH 7) agar plates containing 25 μg/ml of ampicillin wereobtained. Plasmid DNAs were prepared from the transformants of 18clones, and a plasmid DNA which provided a fragment of about 1550 bp bythe EcoRI digestion, which fragment was not digested with BglII(pCRTMIIpurF′ #14) was selected from the plasmid DNAs. The purFcontained in this plasmid DNA has a frame shift at the BglII site, andtherefore it is predicted that the encoded enzyme lacks its function(FIG. 2).

Then, the pCRTMIIpurF′ #14 was digested with EcoRI to prepare a fragmentof about 1.6 Kb that included the purF. This fragment was inserted intothe EcoRI site of pMAN997, which is a vector for homologousrecombination having a temperature-sensitive replication origin (tsori)(As shown in FIG. 1, pMAN031 (J. Bacteriol., 162, 1196 (1985)) of whichVspI-HindIII fragment is replaced with that of pUC19 (Takara Shuzo)), toobtain plasmid pMAN997purF′ #14. E. coli W3110 (wild type) wastransformed at 30° C. with the pMAN997purF′ #14, and some of theobtained colonies were streaked on LB agar plates containing 25 μg/ml ofampicillin, and cultured at 30° C. overnight. Then, the culturedbacterial cells were plated on LB agar plates containing 25 μg/ml ofampicillin so that single colonies should be obtained, to obtaincolonies grown at 42° C. The procedure for obtaining single coloniesgrown at 42° C. was repeated once, and clones in which the whole plasmidwas integrated into the chromosome through homologous recombination wereselected. It was confirmed that these clones did not have the plasmid intheir cytoplasm. Then, several clones among these clones were streakedon LB agar plates, cultured at 30° C. overnight, then inoculated into LBliquid medium (3 ml/test tube), and cultured at 42° C. for 3 to 4 hourswith shaking. The culture broth was appropriately diluted so that singlecolonies should be obtained (about 10⁻⁵ to 10⁻⁶ dilution), plated on LBagar plates, and cultured at 42° C. overnight to obtain colonies. Onehundred colonies were randomly picked up from the emerged colonies, andeach allowed to grow on LB agar plates and LB agar plates containing 25μg/ml of ampicillin, respectively, and ampicillin-sensitive clones grownonly on the LB agar plates were selected. Among the ampicillin-sensitiveclones, clones that were not grown in a minimal medium (Na₂HPO₄ 6.8 g,KH₂PO₄ 3 g, NaCl 0.5 g, NH₄Cl 1 g, MgSO₄.7H₂O 0.5 g, CaCl₂.2H₂O 15 mg,thiamin.HCl 2 mg, glucose 0.2 g per 1 L), but grown in the minimalmedium supplemented with 50 mg/L of hypoxanthine were further selected.Furthermore, the fragment of about 1.5 kb including purF was amplifiedby PCR from the chromosome DNA of the above obtained target clones, andconfirmed not to be digested with BglII. The clones satisfying the aboveconditions were considered strains deficient in purF, and designated asstrains F-2-51 and F-1-72.

2) Breeding of Strain Deficient in Succinyl-AMP Synthase Gene (purA)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, PerkinElmer) by using thechromosome DNA of the strain W3110 as a template and 31-mer primers forboth ends, having nucleotide sequences ofCTCGAGCTCATGGGTAACAACGTCGTCGTAC (SEQ ID NO: 3) andCTCGTCGACTTACGCGTCGAACGGGTCGCGC (SEQ ID NO: 4), and prepared based onthe information of a gene data bank (GenBank Accession No. J04199), andan amplified fragment of about 1300 bp of the purA structural generegion covering ATG and the translation termination codon was clonedbetween the SacI and SalI sites of pUC18 vector (Takara Shuzo). A SacIsite and a SalI site are respectively provided in the PCR primers. Thecloned purA fragment of about 1300 bp contained one HpaI site and oneSnaBI site respectively at about 520 bp and 710 bp from the 5′ end, andtherefore the plasmid was digested with HpaI and SnaBI, and ligated byT4 DNA ligase to obtain the plasmid from which a fragment of about 190bp was removed. Competent cells of E. coli JM109 were transformed withthis ligation solution, and transformants grown on LB agar platescontaining 25 μg/ml of ampicillin were obtained. Plasmid DNAs wereprepared from the transformants of 18 clones, and a plasmid DNA that wasnot digested with FspI but provided a fragment of about 1100 bp fragmentby SacI and SalI digestion (pUC18purA′ #1) was selected from the plasmidDNAs. The purA contained in this plasmid DNA has a deletion between theHpaI and SnaBI sites, and therefore it is predicted that the encodedenzyme lacks its function (FIG. 2).

Then, the pUC18purA′ #1 was digested with SacI and SalI to prepare afragment of about 1.1 kb that included the purA This fragment wasinserted between the SacI and SalI sites of pMAN997, which is a vectorfor homologous recombination having a temperature-sensitive replicationorigin (tsori) (described above), to obtain plasmid pMAN997purA′ #1. Thestrain F-2-51 (purF⁻) was transformed at 30° C. with the plasmidpMAN997purA′ #1, and some of the obtained colonies were streaked on LBagar plates containing 25 μg/ml of ampicillin, and cultured at 30° C.overnight. Then, the cultured bacterial cells were plated on LB agarplates containing 25 μg/ml of ampicillin so that single colonies shouldbe obtained, to obtain colonies grown at 42° C. The procedure forobtaining single colonies grown at 42° C. was repeated once, and clonesin which the whole plasmid was integrated into the chromosome throughhomologous recombination were selected. It was confirmed that theseclones did not have the plasmid in their cytoplasm. Then, several clonesamong these clones were streaked on LB agar plates, cultured at 30° C.overnight, then inoculated into LB liquid medium (3 ml/test tube), andcultured at 42° C. for 3 to 4 hours with shaking The culture broth wasappropriately diluted so that single colonies should be obtained (about10⁻⁵ to 10⁻⁶ dilution), plated on LB agar plates, and cultured at 42° C.overnight to obtain colonies. One hundred colonies were randomly pickedup from the emerged colonies, and each allowed to grow on LB agar platesand LB agar plates containing 25 μg/ml of ampicillin, respectively, andampicillin-sensitive clones grown only on the LB agar plates wereselected. Among the ampicillin-sensitive clones, clones that were notgrown on the minimal medium supplemented with 50 mg/L of hypoxanthine,but grown on the minimal medium supplemented with 50 mg/L of adeninewere further selected. Furthermore, the purA fragment of about 1.1 kbwas amplified by PCR from the chromosome DNA of these target clones, andconfirmed to be smaller than the wild type (about 1.3 kb) and not to bedigested with FspI. The clone satisfying the above conditions wasconsidered a strain deficient in purA, and designated as strain FA-31.

3) Breeding of Strain Deficient in Purine Nucleoside Phosphorylase Gene(deoD)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, Perkin Elmer) by using thechromosome DNA of the strain W3110 as a template and 30-mer and 31-merprimers for both ends, having nucleotide sequences ofCTCGTCGACGCGGGTCTGGAACTGTTCGAC (SEQ ID NO: 5) andCTCGCATGCCCGTGCTTTACCAAAGCGAATC (SEQ ID NO: 6), and prepared based onthe information obtained through searching of a gene data bank (E. coliGene Bank) using “deoD” as a key word, and an amplified fragment ofabout 1350 bp including a deoD structural gene region covering SD-ATGand the translation termination codon was cloned into pCRTMII vector(Invitrogen). The vector has EcoRI sites as restriction sites atvicinities of the both sides of the cloning site. A SalI site and a SphIsite are respectively provided in the PCR primers. The cloned deoDfragment of about 1350 bp contained one HpaI site at about 680 bp fromthe 5′ end, and therefore the plasmid was digested with HpaI, and amixture of the digested plasmid and a 10-mer ClaI linker was subjectedto T4 DNA ligase reaction. As a result, a ClaI site was inserted at theHpaI site. Competent cells of E. coli HB101 were transformed with thisligation solution, and transformants grown on LB agar plates containing25 μg/ml of ampicillin were obtained. Plasmid DNAs were prepared fromthe transformants of 16 clones, and a plasmid DNA that was not digestedwith HpaI but digested with ClaI (pCRTMIIdeoD′ #16) was selected fromthe plasmid DNAs. The deoD contained in this plasmid has a frame shiftat the HpaI site, and therefore it is predicted that the encoded enzymelacks its function (FIG. 2).

Then, the pCRTMIIdeoD′ #16 was digested with EcoRI to prepare a fragmentof about 1.35 kb that included the deoD. This fragment was inserted intothe EcoRI site of pMAN997, which is a vector for homologousrecombination having a temperature-sensitive replication origin (tsori)(described above), to obtain plasmid pMAN997deoD′ #16. The strain F-1-72(purF⁻) and the strain FA-31 (purF⁻, purA⁻) were transformed at 30° C.with plasmid pMAN997deoD′ #16, and some of the obtained colonies werestreaked on LB agar plates containing 25 μg/ml of ampicillin, andcultured at 30° C. overnight. Then, the cultured bacterial cells wereplated on LB agar plates containing 25 μg/ml of ampicillin so thatsingle colonies should be obtained, to obtain colonies grown at 42° C.The procedure for obtaining single colonies grown at 42° C. was repeatedonce, and clones in which the whole plasmid was integrated into thechromosome through homologous recombination were selected. It wasconfirmed that these clones did not have the plasmid in their cytoplasm.Then, several clones among these clones were streaked on LB agar plates,cultured at 30° C. overnight, then inoculated into LB liquid medium (3ml/test tube), and cultured at 42° C. for 3 to 4 hours with shaking. Theculture broth was appropriately diluted so that single colonies shouldbe obtained (about 10⁻⁵ to 10⁻⁶ dilution), plated on LB agar plates, andcultured at 42° C. overnight to obtain colonies. One hundred colonieswere randomly picked up from the emerged colonies, and each allowed togrow on LB agar plates and LB agar plates containing 25 μg/ml ofampicillin, respectively, and ampicillin-sensitive clones grown only onthe LB agar plates were selected. The ampicillin-sensitive clones wereallowed to grow on the LB medium supplemented with 1 g/L of inosine, andclones that did not decompose inosine to hypoxanthine were selectedthrough thin layer chromatography analysis of the culture medium.Furthermore, the fragment of about 1.35 kb including deoD was amplifiedby PCR from the chromosome DNA of these target clones, and confirmed tobe digested with ClaI but not to be digested with HpaI. The clonessatisfying the above conditions were considered strains deficient indeoD, and clones derived from the strain F-1-72 (purF⁻) and the strainFA-31 (purF⁻, purA⁻) were designated as strains FD-6 and FAD-25,respectively.

4) Breeding of Strain Deficient in Purine Repressor Gene (purR)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, PerkinElmer) by using thechromosome DNA of the strain W3110 as a template and 29-mer and 28-merprimers for both ends, having nucleotide sequences ofCTCGTCGACGAAAGTAGAAGCGTCATCAG (SEQ ID NO: 7) andCTCGCATGCTTAACGACGATAGTCGCGG (SEQ ID NO: 8), and prepared based on theinformation obtained through searching of a gene data bank (E. coli GeneBank) using “purR” as a key word, and an amplified fragment of about 1.8kb including a purR structural gene region covering ATG and thetranslation termination codon and about 800 bp 5′ upstream region of ATGwas cloned between the SalI site and the SphI site of pUC19 vector(Takara Shuzo). A SalI site and a SphI site are respectively provided inthe PCR primers, and these sites are used for cloning. The cloned purRfragment of about 1.8 kb contained one PmaCI site at about 810 bp fromthe 5′ end (vicinity of N-terminus in the purR structural gene region),and therefore the plasmid was digested with PmaCI. A mixture of thedigested plasmid and a 8-mer BglII linker was subjected to T4 DNA ligasereaction. As a result, a BglII site was inserted at the PmaCI site.Competent cells of E. coli JM109 were transformed with this ligationsolution, and transformants grown on LB agar plates containing 25 μg/mlof ampicillin were obtained. Plasmid DNAs were prepared from thetransformants of 10 clones, and a plasmid DNA not digested with PmaCIbut digested with BglII (pUC19purR′ #2) was selected from the plasmidDNAs. The purR contained in this plasmid DNA has a frame shift at thePmaCI site, and therefore it is predicted that the encoded enzyme lacksits function (FIG. 2).

Then, the pUC19purR′ #2 was digested with SacI and SphI to prepare afragment of about 1.8 kb that included the purR. This fragment wasinserted between the SacI site and the SphI site of pMAN997, which is avector for homologous recombination having a temperature-sensitivereplication origin (tsori) (described above), to obtain plasmidpMAN997purR′ #2. The strain FD-6 (purF⁻, deoD⁻) and the strain FAD-25(purF⁻, purA⁻, deoD⁻) were transformed at 30° C. with the plasmidpMAN997purR′ #2, and some of the obtained colonies were streaked on LBagar plates containing 25 μg/ml of ampicillin, and cultured at 30° C.overnight. Then, the cultured bacterial cells were plated on LB agarplates containing 25 μg/ml of ampicillin so that single colonies shouldbe obtained, to obtain colonies grown at 42° C. The procedure forobtaining single colonies grown at 42° C. was repeated once, and clonesin which the whole plasmid was integrated into the chromosome throughhomologous recombination were selected. It was confirmed that theseclones did not have the plasmid in their cytoplasm. Then, several clonesamong these clones were streaked on LB agar plates, cultured at 30° C.overnight, then inoculated into LB liquid medium (3 ml/test tube), andcultured at 42° C. for 3 to 4 hours with shaking. The culture broth wasappropriately diluted so that single colonies should be obtained (about10⁻⁵ to 10⁻⁶ dilution), plated on LB agar plates, and cultured at 42° C.overnight to obtain colonies. One hundred colonies were randomly pickedup from the emerged colonies, and each allowed to grow on LB agar platesand LB agar plates containing 25 μg/ml of ampicillin, respectively, andampicillin-sensitive clones grown only on the LB agar plates wereselected. Ten clones were randomly selected from theampicillin-sensitive clones, and the fragment of about 1.8 kb includingpurR was amplified by PCR from the chromosome DNA of these clones, andclones that were digested with BglII but not with PmaCI were selected.These clones were considered strains deficient in purR, and clonesderived from the strain FD-6 (purr, deoD⁻) and the strain FAD-25 (purF⁻,purA⁻, deoD⁻) are designated as strain FDR-18 and strain FADR-8,respectively. It was confirmed that the PRPP amidotransferase activityin the strains in which purR was destroyed was increased compared withthat of a strain in which purR was not destroyed by using the purF⁺strain deficient in deoD and purR or the purF⁺ strain deficient in purA,deoD and purR. The PRPP amidotransferase activity was measured accordingto the method of L. J. Messenger et al. (J. Biol. Chem., 254, 3382(1979)).

5) Construction of Desensitized Type PRPP Amidotransferase Gene (purF)

A purF fragment was excised from the plasmid carrying the purF of about1530 bp cloned into pCRTMII vector (Invitrogen) in Section 1) bydigestion with PstI and HindIII, and inserted between the PstI andHindIII sites of the multi-cloning site of a plasmid for introducingmutation, pKF18 (Takara Shuzo) to obtain the target clone (pKFpurF). G.Zhou et al. (J. Biol. Chem., 269, 6784 (1994)) has revealed that PRPPamidotransferase (PurF) whose Lys (K) at position 326 is replaced withGln (Q) and the same whose Pro (P) at position 410 is further replacedwith Trp (W) are each desensitized for feedback inhibition by GMP andAMP. Therefore, the following synthetic DNA primers were prepared forgene substitution realizing mutations of Lys (K) at position 326 and Pro(P) at position 410 of PRPP amidotransferase (PurF) to Gln (Q) and Trp(W), respectively, and pKFpurF was subjected to site-directedmutagenesis according to the protocol of Site-directed MutagenesisSystem Mutan-Super Express Km (Takara Shuzo) to introduce asite-directed mutation into the pKFpurF. Primer for K326Q mutation: (SEQID NO: 9) 5′-GGGCTTCGTT CAG AACCGCTATGTTGG-3′ Primer for P410W mutation:(SEQ ID NO: 10) 5′-TATGGTATTGATATG TGG AGCGCCACGGAAC-3′

After the mutagenesis, 6 clones were randomly picked up from each of theresulting transformants, and plasmids were prepared from them. Bynucleotide sequencing of the plasmids around the locations where themutations were introduced, it was confirmed that target mutants wereobtained. The obtained plasmids were designated as pKFpurFKQ andpKFpurFPW, respectively. The mutation P410 W (410Pro→Trp) was furtherintroduced into pKFpurFKQ in the same manner to prepare pKFpurFKQPW, amutant plasmid having two mutations simultaneously. Each of the plasmidspKFpurFKQ, pKFpurFPW and pKFpurFKQPW has an inserted mutant purFdownstream of the lacp/o (promoter of lactose operon) derived frompKF18, and the purF is expressed under the control of this promoter.

Recombinant bacteria obtained by transforming E. coli JM109 cells withthe above plasmids were cultured in LB liquid medium for eight hours,and collected, and crude enzyme extracts were prepared from them. ThePRPP amidotransferase activity of the extracts and degrees of inhibitionby AMP and GMP were measured according to the method of L. J. Messenger(J. Biol. Chem., 254, 3382 (1979)). The results are shown in Table 1.TABLE 1 PRPP amidotransferase activity and inhibition by AMP and GMPPRPP amidotransferase activity (μmole/min/mg) Host Plasmid None 10 mMAMP 10 mM GMP JM109 — 0.001 — — JM109 pKFpurF 0.68 0.48 0.10 JM109pKFpurFKQ 0.34 0.32 0.33 JM109 pKFpurFKQPW 0.18 0.16 0.17

6) Evaluation of Purine Nucleoside-Producing Ability of Mutant purFPlasmid-Introduced Strain

Transformants were produced by introducing pKFpurFKQ and pKFpurFKQPWinto the strain FDR-18 (purF⁻, deoD⁻, purR⁻) and the strain FADR-8(purF⁻, purA⁻, deoD⁻, purR⁻) produced in Section 4), and purinenucleoside-producing abilities of these strains were evaluated.

Basal medium and culture method for purine nucleoside production andanalysis method for the evaluation of the purine nucleoside-producingability will be described below. 1. Basal medium: MS medium Finalconcentration Glucose 40 g/L (separately sterilized) (NH₄)₂SO₄ 16 g/LKH₂PO₄ 1 g/L MgSO₄•7H₂O 1 g/L FeSO₄•7H₂O 0.01 g/L MnSO₄•4H₂O 0.01 g/LYeast extract 2 g/L CaCO₃ 30 g/L (separately sterilized)2. Culture Method

-   Refresh culture; a stored bacterium inoculated LB agar medium    (supplemented with a drug as required) 37° C., cultured overnight-   Seed culture; the refresh cultured bacterium inoculated LB liquid    medium (supplemented with a drug as required) 37° C., cultured    overnight-   Main culture; 2% inoculated from the seed culture-   MS medium (supplemented with adenine and a drug as required)-   37° C., 20 ml/500-ml volume Sakaguchi's culture flask    3. Analysis Method

A sample of the culture medium (500 μl) is repeatedly taken in the timecourse, and centrifuged at 15,000 rpm for 5 minutes, and the supernatantis diluted 4 times with H₂O and analyzed by HPLC. Unless notedotherwise, the evaluation is made based on an accumulated amount of apurine nucleoside per unit volume of the medium after culture for 3days.

Analysis Conditions:

Column: Asahipak GS-220 (7.6 mm ID×500 mm L)

Buffer: pH is adjusted with 0.2M NaH₂PO₄ (pH 3.98), and phosphoric acid

Temperature: 55° C.

Flow Rate: 1.5 ml/min

Detection: UV 254 nm Retention time (min) Inosine 16.40 Hypoxanthine19.27 Guanosine 20.94 Guanine 23.55 Adenine 24.92 Adenosine 26.75

For the strains of purA⁻ (adenine auxotrophic), 5 mg/L of adenine wasadded to the MS medium.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 2. Superior inosine production was observed withrespect to the mutant purF plasmid-introduced strains in contrast to thestrain W3110 (wild type strain) by which a trace amount of theproduction was observed. TABLE 2 Evaluation of purinenucleoside-producing ability Purine nucleoside accumulation InosineGuanosine Host Plasmid (mg/L) (mg/L) W3110 — Trace 0 FDR-18 pKFpurFKQ115 0 FDR-18 pKFpurFKQPW 110 0 FADR-8 pKFpurFKQ 66 0 FADR-8 pKFpurFKQPW62 0

Example 2 1) Breeding of Strain Deficient in Adenosine Deaminase Gene(add)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, Perkin Elmer) by using thechromosome DNA of the strain W3110 as a template and 29-mer primers forboth ends, having nucleotide sequences of CTCGTCGACGGCTGGATGCCTTACGCATC(SEQ ID NO: 11) and CTCGCATGCAGTCAGCACGGTATATCGTG (SEQ ID NO: 12), andprepared based on the information obtained through searching of a genedata bank (E. coli Gene Bank) using “add” as a key word, and anamplified fragment of about 1.8 kb including an add structural generegion covering ATG and the translation termination codon, about 420 bp5′ upstream region of ATG and about 370 bp downstream region of thetranslation termination codon was cloned between the SalI site and theSphI site of pUC19 vector (Takara Shuzo). A SalI site and a SphI siteare respectively provided in the PCR primers, and these sites are usedfor the cloning. The cloned add fragment of about 1.8 kb contained oneStuI site at about 880 bp from the 5′ end, and therefore the plasmid wasdigested with StuI, and a mixture of the digested plasmid and a 8-merBglII linker was subjected to T4 DNA ligase reaction. As a result, aBglII site was inserted at the StuI site. Competent cells of E. coliJM109 were transformed with this ligation solution, and transformantsgrown on LB agar plates containing 25 μg/ml of ampicillin were obtained.Plasmid DNAs were prepared from the transformants of 10 clones, and aplasmid DNA not digested with StuI but digested with BglII (pUC19add′#1) was selected from the plasmid DNAs. The add contained in thisplasmid DNA has a frame shift at the StuI site, and therefore it ispredicted that the encoded enzyme lacks its function (FIG. 2).

Then, the pUC19add′ #1 was digested with SacI and SphI to prepare afragment of about 1.8 kb that included the add. This fragment wasinserted between the SacI site and the SphI site of pMAN997, which is avector for homologous recombination having a temperature-sensitivereplication origin (tsori) (described above), to obtain plasmidpMAN997add′ #1. The strain FDR-18 (purF⁻, deoD⁻, purR⁻) and the strainFADR-8 (purF⁻, purA⁻, deoD⁻, purR⁻) were transformed at 30° C. with theplasmid pMAN997add′ #1, and some of the obtained colonies were streakedon LB agar plates containing 25 μg/ml of ampicillin, and cultured at 30°C. overnight. Then, the cultured bacterial cells were plated on LB agarplates containing 25 μg/ml of ampicillin so that single colonies shouldbe obtained, to obtain colonies grown at 42° C. The procedure forobtaining single colonies grown at 42° C. was repeated once, and clonesin which the whole plasmid was integrated into the chromosome throughhomologous recombination were selected. It was confirmed that theseclones did not have the plasmid in their cytoplasm. Then, several clonesamong these clones were streaked on LB agar plates, cultured at 30° C.overnight, then inoculated into LB liquid medium (3 ml/test tube), andcultured at 42° C. for 3 to 4 hours with shaking. The culture broth wasappropriately diluted so that single colonies should be obtained (about10⁻⁵ to 10⁻⁶ dilution), plated on LB agar plates, and cultured at 42° C.overnight to obtain colonies. One hundred colonies were randomly pickedup from the emerged colonies, and each allowed to grow on LB agar platesand LB agar plates containing 25 μg/ml of ampicillin, respectively, andampicillin-sensitive clones grown only on the LB agar plates wereselected. The ampicillin-sensitive clones were allowed to grow in the LBmedium supplemented with 1.5 g/L of adenosine, and clones that did notconvert adenosine to inosine were selected through thin layerchromatography analysis of the culture medium. Furthermore, the addfragment of about 1.8 kb was amplified by PCR from the chromosome DNA ofthese target clones, and confirmed to be digested with BglII but not tobe digested with StuI. These clones were considered strains deficient inadd, and clones derived from the strain FDR-18 (purF⁻, deoD⁻, purR⁻) andthe strain FADR-8 (purF⁻, purA⁻, deoD⁻, purR⁻) are designated as strainsFDRadd-18-1 and FADRadd-8-3, respectively.

2) Evaluation of Purine Nucleoside-Producing Ability of DesensitizedType purF-Introduced Strain

Transformants were made by introducing pKFpurFKQ and pKFpurFKQPW intothe strain FDRadd-18-1 (purF⁻, deoD⁻, purR, add⁻) and the strainFADRadd-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻) bred in Section 1), andpurine nucleoside-producing abilities of these strains were evaluated.For the strain FADRadd-8-3, a transformant with the wild type purFplasmid (pKFpurF) was also made, and compared with the transformant withpKFpurFKQ and the transformant with pKFpurFKQPW. The basal medium andthe culture method for the purine nucleoside production and the analysismethod were the same as Example 1.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 3. Superior inosine production was observed comparedwith the strain W3110 (wild type strain). Effects of desensitized typepurFKQ and purFKQPW were observed by comparing with the wild type purF.TABLE 3 Evaluation of purine nucleoside-producing ability Purinenucleoside accumulation Inosine Guanosine Host Plasmid (mg/L) (mg/L)W3110 — Trace 0 FDRadd-18-1 pKFpurFKQ 220 0 FDRadd-18-1 pKFpurFKQPW 2150 FADRadd-8-3 pKFpurFKQ 1080 0 FADRadd-8-3 pKFpurFKQPW 1030 0FADRadd-8-3 pKFpurF 805 0

Example 3 1) Construction of Desensitized Type purF Plasmid forHomologous Recombination

In order to introduce desensitized type purF substitution in achromosome by using the purF strain produced in Example 1, Section 1),another purF fragment longer than the previously obtained purF fragment(about 1.6 kb) by about 0.5 kb for the 3 side was prepared. PCR wascarried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30 cycles;Gene Amp PCR System Model 9600, Perkin Elmer) by using the chromosomeDNA of the strain W3110 and 29-mer primers for both ends, havingnucleotide sequences of CTCCTGCAGAACGAGGAAAAAGACGTATG (SEQ ID NO: 1) andCTCAAGCTTGTCTGATTTATCACATCATC (SEQ ID NO: 13), and prepared based on theinformation of the gene data bank (E. coli Gene Bank), and an amplifiedfragment of about 2.1 kb including the purF structural gene regioncovering SD-ATG and the translation termination codon was cloned intopCRTMII vector (Invitrogen). The plasmid contained in this clone isdesignated as pCRTMIIpurFL. The pCRTMIIpurFL has EcoRI sites asrestriction sites at vicinities of the both sides of the cloning site. APstI site and a HindIII site are respectively provided in the PCRprimers.

Then, the pCRTMIIpurFL was digested with SnaBI and HindIII to obtain afragment of about 0.65 kb present downstream of the C-terminus of thepurF coding region. This fragment was inserted between the SnaBI siteand the HindIII site of pKFpurFKQ and pKFpurFKQPW obtained in Example 1,Section 5) to obtain pKFpurFLKQ and pKFpurFLKQPW.

Then, the pKFpurFLKQ and pKFpurFLKQPW were digested with EcoRI andHindIII to give fragments of about 2.1 kb containing purFLKQ andpurFLKQPW. These fragments were inserted between the EcoRI and HindIIIsites of pMAN997, which is a vector for homologous recombination havinga temperature-sensitive replication origin (tsori) (described above), toobtain plasmids pMAN997purFLKQ and pMAN997purFLKQPW, respectively.

2) Breeding of Strain Having Desensitized Type purF Integrated inChromosome

The strain FDRadd-18-1 (purF⁻, deoD⁻, purR⁻, add⁻) and the strainFADRadd-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻) were each transformedwith the plasmids pMAN997purFLKQ and pMAN997purFLKQPW at 30° C., andsome of the obtained colonies were streaked on LB agar plates containing25 μg/ml of ampicillin, and cultured at 30° C. overnight. Then, thecultured bacterial cells were plated on LB agar plates containing 25μg/ml of ampicillin so that single colonies should be obtained, toobtain colonies grown at 42° C. The procedure for obtaining singlecolonies grown at 42° C. was repeated once, and clones in which thewhole plasmid was integrated into the chromosome through homologousrecombination were selected. It was confirmed that these clones did nothave the plasmid in their cytoplasm. Then, several clones among theseclones were streaked on LB agar plates, cultured at 30° C. overnight,then inoculated into LB liquid medium (3 ml/test tube), and cultured at42° C. for 3 to 4 hours with shaking. The culture was appropriatelydiluted so that single colonies should be obtained (about 0-5 to 10⁻⁶dilution), plated on LB agar plates, and cultured at 42° C. overnight toobtain colonies. One hundred colonies were randomly picked up from theemerged colonies, and each allowed to grow on LB agar plates and LB agarplates containing 25 μg/ml of ampicillin, respectively, andampicillin-sensitive clones grown only on the LB agar plates wereselected. Among the ampicillin-sensitive clones, clones that were grownon the minimal medium were selected for the strain FDRadd-18-1 (purF⁻,deoD⁻, purR⁻, add⁻), and clones that were grown in the minimal mediumsupplemented with 100 mg/L of L-histidine and 50 mg/L of adenine wereselected for the strain FADRadd-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻).

The fragments of about 1.5 kb including purF were amplified fromchromosome DNA of these target clones, and nucleotide sequences aroundthe locations where mutations were introduced by homologousrecombination substitution were determined. As a result, it wasconfirmed that they contained the mutation of K326Q (326Lys→Gln), andthe mutations of K326Q (326Lys→Gln)+P410 W (410Pro→Trp), respectively.

Those derived from the strain FDRadd-18-1 (purF⁻, deoD⁻, purR⁻, add⁻)were designated as strain FDRadd-18-1::KQ (purFKQ, deoD⁻, purR⁻, add⁻)and strain FDRadd-18-1::KQPW (purFKQPw, deoD⁻, purR⁻, add⁻), and thosederived from the strain FADRadd-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻)were designated as strain FADRadd-8-3::KQ (purFKQ, purA⁻, deoD⁻, purR⁻,add⁻) and strain FADRadd-8-3::KQPW (purFKQPW, purA⁻, deoD⁻, purR⁻,add⁻).

3) Evaluation of Purine Nucleoside-Producing Ability of Strain HavingDesensitized Type purF Integrated into Chromosome

Purine nucleoside-producing abilities of the strain FDRadd-18-1::KQ(purFKQ, deoD⁻, purR⁻, add⁻), the strain FDRadd-18-1::KQPW (purFKQPW,deoD⁻, purR⁻, add⁻), the strain FADRadd-8-3::KQ (purFKQ, purA⁻, deoD⁻,purR⁻, add⁻) and the strain FADRadd-8-3::KQPW (purFKQPW, purA⁻, deoD⁻,purR⁻, add⁻) prepared in Section 2) were evaluated. The basal medium andthe culture method for the purine nucleoside production and the analysismethod were the same as Example 1. For the strains of purA⁻ (adenineauxotrophic), 5 mg/L of adenine was added to the MS medium.

The results of the evaluation of purine nucleoside-producing ability areshown in Table 4. Superior inosine production was observed compared withthe strain W3110 (wild type strain). TABLE 4 Evaluation of purinenucleoside-producing ability Purine nucleoside accumulation InosineGuanosine Strain (mg/L) (mg/L) W3110 Trace 0 FDRadd-18-1::KQ 110 0FDRadd-18-1::KQPW 105 0 FADRadd-8-3::KQ 635 0 FADRadd-8-3::KQPW 620 0

Example 4 1) Breeding of Strain Deficient in Inosine-Guanosine KinaseGene (gsk)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, Perkin Elmer) by using thechromosome DNA of the strain W3110 and 23-mer and 21-mer primers forboth ends, having nucleotide sequences of CTCGAGCTCATGAAATTTCCCGG (SEQID NO: 14) and CTCGGATCCGGTACCATGCTG (SEQ ID NO: 15), and prepared basedon the information of a gene data bank (GenBank Accession No. D00798),and an amplified fragment of about 1.5 kb including the gsk structuralgene region covering ATG and the translation termination codon wascloned between the SacI site and the BamHI site of pUC18 vector (TakaraShuzo). A SacI site and a BamHI site are respectively provided in thePCR primers.

The cloned gsk fragment of 1.5 kb contained one BglII site at about 830bp from the 5′ end, and therefore the plasmid was digested with BglII,and subjected to T4 DNA ligase reaction in order to insert a kanamycinresistant (Km^(r)) gene GenBlock (BamHI digest, Pharmacia Biotech).Competent cells of E. coli JM109 were transformed with this ligationsolution, and transformants grown on LB agar plates containing 50 μg/mlof kanamycin were obtained. Plasmid DNAs were prepared from thetransformants of 4 clones, and a plasmid DNA that was not digested withBglII from which plasmid a fragment of about 2.8 kp was excised by EcoRIand SalI digestion (pUCgsk′ #2) was selected from the plasmid DNAs. Thegsk contained in this plasmid has an inserted heterogeneous gene at theBglII site, and therefore it is predicted that the encoded enzyme lacksits function (FIG. 2).

Then, the pUCgsk′ #2 was digested with SacI, SphI and DraI to prepare afragment of about 2.8 Kb that included the gsk and Km^(r) genes. TheDraI digestion is employed to facilitate the preparation of a SacI-SphIfragment. The fragment was inserted between the SacI and SphI sites ofpMAN997, which is a vector for homologous recombination having atemperature-sensitive replication origin (tsori) (describe above), toobtain plasmid pMAN997gsk′ #2. The strain FDR-18 (purF⁻, deoD⁻, purR⁻)and the strain FADRadd-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻) weretransformed at 30° C. with the plasmid pMAN997gsk′ #2, and some of theobtained colonies were streaked on LB agar plates containing 25 μg/ml ofampicillin, and cultured at 30° C. overnight. Then, the culturedbacterial cells were plated on LB agar plates containing 25 μg/ml ofampicillin so that single colonies should be obtained, to obtaincolonies grown at 42° C. The procedure for obtaining single coloniesgrown at 42° C. was repeated once, and clones in which the whole plasmidwas integrated into the chromosome through homologous recombination wereselected. It was confirmed that these clones did not have the plasmid intheir cytoplasm. Then, several clones among these clones were streakedon LB agar plates, cultured at 30° C. overnight, then inoculated into LBliquid medium (3 ml/test tube), and cultured at 42° C. for 3 to 4 hourswith shaking. The culture was appropriately diluted so that singlecolonies should be obtained (about 10⁻⁵ to 10⁻⁶ dilution), plated on LBagar plates, and cultured at 42° C. overnight to obtain colonies. Onehundred colonies were randomly picked up from the emerged colonies, andeach allowed to grow on LB agar plates, LB agar plates containing 25μg/ml of ampicillin, respectively, and LB agar plates containing 20μg/ml of kanamycin, respectively, and clones not grown on the LB agarplates containing 25 μg/ml of ampicillin, but grown on the LB agarplates containing 20 μg/ml of kanamycin were selected. Furthermore, thefragment including the gsk gene was amplified by PCR from the chromosomeDNA of these target clones, and it was confirmed that the about 2.8 kbfragment including Km^(r) gene, not the original fragment of about 1.5kb, was amplified. It was also confirmed that the inosine-guanosinekinase activity was not detected in them. The inosine-guanosine kinaseactivity was measured according to the method of Usuda et al. (Biochim.Biophys. Acta., 1341, 200-206 (1997)). Those clones were consideredstrains deficient in gsk, and clones derived from the strain FDR-18(purF⁻, deoD⁻, purR⁻) and the strain FADRadd-8-3 (purF⁻, purA⁻, deoD⁻,purR⁻, add⁻) are designated as strain FDRG-18-13 and strainFADRaddG-8-3, respectively.

2) Evaluation of Purine Nucleoside-Producing Ability of DesensitizedType purF Plasmid-Introduced Strain

Because the plasmids pKFpurFKQ and pKFpurFKQPW have the Km^(r) gene as adrug selection marker and the host strains FDRG-18-13 (purF⁻, deoD⁻,purR⁻, gsk⁻) and FADRaddG-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, gsk⁻)prepared in Section 1) are made kanamycin resistant, it is difficult toobtain transformants by introducing pKFpurFKQ and pKFpurFKQPW into thestrains FDRG-18-13 and FADRaddG-8-3 for the evaluation of the purinenucleoside-producing ability. Therefore, exchange of drug selectionmarker genes of the plasmids pKFpurFKQ and pKFpurFKQPW was performed byusing pUC18 vector having the ampicillin resistance gene (Takara Shuzo).Because the locational relationship between the lac promoter and themulti-cloning site is common to pKF18 and pUC18, purFKQ and purFKQPWfragments were excised from pKFpurFKQ and pKFpurFKQPW by using PstI andHindIII, and these were inserted between the PstI and HindIII sites ofpUC18 to prepare pUCpurFKQ and pUCpurFKQPW. The hosts, the strainsFDRG-18-13 and FADRaddG-8-3, were transformed with the pUCpurFKQ andpUCpurFKQPW, and the purine nucleoside-producing abilities of therecombinants were evaluated. The basal medium and the culture method forthe purine nucleoside production and the analysis method were the sameas Example 1. For the strains of purA⁻ (adenine auxotrophic), 5 mg/L ofadenine was added to the MS medium.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 5. From these results, it was revealed that themicroorganisms accumulated guanosine as well as inosine when thedeficiency of gsk was added. TABLE 5 Evaluation of purinenucleoside-producing ability Purine nucleoside accumulation InosineGuanosine Host Plasmid (mg/L) (mg/L) W3110 — Trace 0 FDRG-18-13pUCpurFKQ 105 139 FDRG-18-13 pUCpurFKQPW 108 93 FADRaddG-8-3 pUCpurFKQ126 52 FADRaddG-8-3 pUCpurFKQPW 222 49

3) Breeding of Strains Having Desensitized Type purF Integrated intoChromosome and Evaluation of Purine Nucleoside-Producing Ability

The strains FDRG-18-3 (purF⁻, deoD⁻, purR⁻, gsk⁻) and FADRaddG-8-3(purr, purA⁻, deoD⁻, purR⁻, add⁻, gsk⁻) were transformed at 30° C. withthe plasmids pMAN997purFLKQ and pMAN997purFLKQPW, respectively. Some ofthe obtained colonies were streaked on LB agar plates containing 25μg/ml of ampicillin, and cultured at 30° C. overnight. Then, thecultured bacterial cells were plated on LB agar plates containing 25μg/ml of ampicillin so that single colonies should be obtained, toobtain colonies grown at 42° C. The procedure for obtaining singlecolonies grown at 42° C. was repeated once, and clones in which thewhole plasmid was integrated into the chromosome through homologousrecombination were selected. It was confirmed that these clones did nothave the plasmid in their cytoplasm. Then, several clones among theseclones were streaked on LB agar plates, cultured at 30° C. overnight,then inoculated into LB liquid medium (3 ml/test tube), and cultured at42° C. for 3 to 4 hours with shaking. The culture was appropriatelydiluted so that single colonies should be obtained (about 10⁻⁵ to 10⁻⁶dilution), plated on LB agar plates, and cultured at 42° C. overnight toobtain colonies. One hundred colonies were randomly picked up from theemerged colonies, and each allowed to grow on LB agar plates and LB agarplates containing 25 μg/ml of ampicillin, respectively, andampicillin-sensitive clones grown only on the LB agar plates wereselected. From ampicillin-sensitive clones, clones grown on the minimalmedium were further selected for the strain FDRG-18-13 (purF⁻, deoD⁻,purR⁻, gsk⁻), and clones grown in the minimal medium supplemented with100 mg/L of L-histidine and 50 mg/L of adenine were selected for thestrain FADRaddG-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, gsk⁻).

Chromosome DNAs of these target clones were prepared, and fragments ofabout 1.5 kb including purF were amplified by PCR, and sequenced aroundthe locations where the mutations were introduced thorough substitutionby homologous recombination. As a result, it was confirmed that they hada mutation of K326Q (326Lys→Gln) and K326Q (326Lys→Gln)+P410 W(410Pro→Trp), respectively.

The strains derived from the strain FDRG-18-13 (purF⁻, deoD⁻, purR⁻,gsk⁻) were designated as strain FDRG-18-13::KQ (purFKQ, deoD⁻, purR⁻,gsk⁻) and strain FDRG-18-13::KQPW (purFKQPW, deoD⁻, purR⁻, gsk⁻), andthose derived from the strain FADRaddG-8-3 (purR⁻, purA⁻, deoD⁻, purR⁻,add⁻, gsk⁻) were designated as strain FADRaddG-8-3::KQ (purFKQ, purA⁻,deoD⁻, purR⁻, add⁻, gsk⁻) and strain FADRaddG-8-3::KQPW (purFKQPW,purA⁻, deoD⁻, purR⁻, add⁻, gsk⁻).

The strain FADRaddG-8-3::KQ (purFKQ, purA⁻, deoD⁻, purR⁻, add⁻, gsk⁻)was given a private number AJ13334. This strain was deposited atNational Institute of Bioscience and Human-Technology of Ministry ofInternational Trade and Industry (1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki 305-0046 Japan) on Jun. 24, 1997 as an international depositionunder the Budapest treaty, and received an accession number FERMBP-5993.

The purine nucleoside-producing abilities of these four kinds of strainshaving desensitized type purF integrated into chromosome were evaluated.The basal medium and the culture method for the purine nucleosideproduction and the analysis method were the same as Example 1. For thestrains of purA⁻ (adenine auxotrophic), 5 mg/L of adenine was added tothe MS medium.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 6. From these results, it was revealed that themicroorganisms accumulated guanosine as well as inosine when thedeficiency of gsk was added. TABLE 6 Evaluation of purinenucleoside-producing ability Purine nucleoside accumulation InosineGuanosine Strain (mg/L) (mg/L) W3110 Trace 0 FDRG-18-13::KQ 150 140FDRG-18-13::KQPW 145 125 FADRaddG-8-3::KQ 550 135 FADRaddG-8-3::KQPW 530130

Example 5 1) Construction of Wild Type purR Plasmid for HomologousRecombination and Breeding of Strain Having Reversed purR⁺ Integratedinto Chromosome

In Example 1, Section 4), the plasmid (pUCpurR) carrying the purRfragment of about 1.8 kb between the SalI site and the SphI site ofpUC19 vector (Takara Shuzo) was obtained. The pUCpurR was digested withSacI and SphI to prepare a fragment of about 1.8 kb that included wildtype purR. This fragment was inserted between the SacI site and the SphIsite of pMAN997, which is a vector for homologous recombination having atemperature-sensitive replication origin (tsori) (described above), toobtain plasmid pMAN997purR. The strain FADRadd-8-3 (purF⁻, purA⁻, deoD⁻,purR⁻, add⁻) was transformed at 30° C. with the plasmid pMAN997purR, andsome of the obtained colonies were streaked on LB agar plates containing25 μg/ml of ampicillin, and cultured at 30° C. overnight. Then, thecultured bacterial cells were plated on LB agar plates containing 25μg/ml of ampicillin so that single colonies should be obtained, toobtain colonies grown at 42° C. The procedure for obtaining singlecolonies grown at 42° C. was repeated once, and clones in which thewhole plasmid was integrated into the chromosome through homologousrecombination were selected. It was confirmed that these clones did nothave the plasmid in their cytoplasm. Then, several clones among theseclones were streaked on LB agar plates, cultured at 30° C. overnight,then inoculated into LB liquid medium (3 ml/test tube), and cultured at42° C. for 3 to 4 hours with shaking. The culture was appropriatelydiluted so that single colonies should be obtained (about 10⁻⁵ to 10⁻⁶dilution), plated on LB agar plates, and cultured at 42° C. overnight toobtain colonies. One hundred colonies were randomly picked up from theemerged colonies, and each allowed to grow on LB agar plates and LB agarplates containing 25 μg/ml of ampicillin, respectively, andampicillin-sensitive clones grown only on the LB agar plates wereselected. 10 clones were randomly selected from the ampicillin-sensitiveclones, and the purR fragments of about 1.8 kb were amplified from thechromosome DNA of these clones by PCR. Clones in which the amplifiedfragment was digested with PmaCI but not with BglII were selected. Theclones were considered purR⁺ reversed strains, and designated asFADadd-8-3-2 (purF⁻, purA⁻, deoD⁻, add⁻).

2) Evaluation of Purine Nucleoside-Producing Ability of DesensitizedType purF-Introduced Strain

A transformant was produced by introducing pKFpurFKQ into the strainFADadd-8-3-2 (purF⁻, purA⁻, deoD⁻, add⁻), and purinenucleoside-producing ability of the strain was evaluated. For the strainFADRadd-8-3, a transformant with pKFpurKQ was also prepared, and aneffect of purR deficiency was evaluated by comparison. The basal mediumand the culture method for the purine nucleoside production and theanalysis method were the same as Example 1. The MS medium wassupplemented with 5 mg/L adenine.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 7. Superior inosine production was observed inFADRadd (purR⁻ type) compared with FADadd (purR wild type) and theeffect of purR deficiency was confirmed. TABLE 7 Evaluation of purinenucleoside-producing ability Purine nucleoside accumulation InosineGuanosine Host Plasmid (mg/L) (mg/L) W3110 — Trace 0 FADRadd-8-3pKFpurFKQ 1080 0 FADadd-8-3-2 pKFpurFKQ 930 0

Example 6 1) Rebreeding of Strain Deficient in Inosine-Guanosine KinaseGene (gsk)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, Perkin Elmer) by using thechromosome DNA of the strain W3110 and 32-mer and 29-mer primers forboth ends, having nucleotide sequences ofCTCGGTACCCTGTTGCGTTAAGCCATCCCAGA (SEQ ID NO: 16) andCTCGCATGCCAACGTACGGCATTAACCTA (SEQ ID NO: 17), and prepared based on theinformation of a gene data bank (GenBank Accession No. D00798), and anamplified fragment of about 3.0 kb including the gsk structural generegion (about 800 bp) covering ATG and the translation termination codonwas cloned between the KpnI site and the SphI site of pUC19 vector(Takara Shuzo). A KpnI site and a SphI site are respectively provided inthe PCR primers.

The cloned gsk fragment of 3.0 kb contained two Aro51HI sites at about900 bp and 1030 bp and one BglII site at about 1640 bp from the 5 end,and therefore the plasmid was digested with Aro51HI and BglII, andblunt-ended by T4 DNA polymerase. Then the Aro51HI-BglII fragment wasremoved and DNA of the vector was subjected to self-ligation by T4 DNAligase. Competent cells of E. coli JM109 were transformed with thisligation solution, and transformants grown on LB agar plates containing25 μg/ml of ampicillin were obtained. Plasmid DNAs were prepared fromthe transformants of 10 clones, and a plasmid DNA which was not digestedwith Aro51HI or BglII and from which plasmid a fragment of about 2.3 kpwas excised by KpnI and SphI digestion (pUC19gsk′ #10) was selected fromthe plasmid DNAs. The gsk contained in this plasmid has a deletion inthe structural gene between the Aro51HI site and the BglII site, andtherefore it is predicted that the encoded enzyme lacks its function(FIG. 3).

Then, the pUC19gsk′ #10 was digested with KpnI and SphI to prepare afragment of about 2.3 kb that included the gsk gene. The fragment wasinserted between the KpnI and SphI sites of pMAN997, which is a vectorfor homologous recombination having a temperature-sensitive replicationorigin (tsori) (describe above), to obtain plasmid pMAN997gsk′ #10. Thestrain FADRadd-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻) was transformed at30° C. with the plasmid pMAN997gsk′ #10, and some of the obtainedcolonies were streaked on LB agar plates containing 25 μg/ml ofampicillin, and cultured at 30° C. overnight. Then, the culturedbacterial cells were plated on LB agar plates containing 25 μg/ml ofampicillin so that single colonies should be obtained, to obtaincolonies grown at 42° C. The procedure for obtaining single coloniesgrown at 42° C. was repeated once, and clones in which the whole plasmidwas integrated into the chromosome through homologous recombination wereselected. It was confirmed that these clones did not have the plasmid intheir cytoplasm. Then, several clones among these clones were streakedon LB agar plates, cultured at 30° C. overnight, then inoculated into LBliquid medium (3 ml/test tube), and cultured at 42° C. for 3 to 4 hourswith shaking. The culture was appropriately diluted so that singlecolonies should be obtained (about 10⁻⁵ to 10⁻⁶ dilution), plated on LBagar plates, and cultured at 42° C. overnight to obtain colonies. Onehundred colonies were randomly picked up from the emerged colonies, andeach allowed to grow on LB agar plates and LB agar plates containing 25μg/ml of ampicillin, respectively, and ampicillin-sensitive clones grownonly on the LB agar plates were selected. Furthermore, 10 clones wererandomly selected from the ampicillin-sensitive clones, the fragmentsincluding the gsk gene were amplified by PCR using the above PCR primersfrom the chromosome DNA of these target clones, and the clones in whichthe fragment of about 2.3 kb, not the original fragment of about 3.0 kbwere amplified were selected. It was also confirmed that theinosine-guanosine kinase activity was not detected in them. Theinosine-guanosine kinase activity was measured according to the methodof Usuda et al. (Biochim. Biophys. Acta., 1341, 200-206 (1997)). Theclones were considered new strains deficient in gsk, and the clonesderived from the strain FADRadd-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻)were designated as FADRaddgsk (purF⁻, purA⁻, deoD⁻, purR⁻, add, gsk).

2) Breeding of Strain Deficient in GMP Reductase Gene (guaC)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, Perkin Elmer) by using thechromosome DNA of the strain W3110 and 29-mer primers for both ends,having nucleotide sequences of CTCAAGCTTACGGCTCTGGTCCACGCCAG (SEQ ID NO:18) and CTCCTGCAGCAGCGTTGGGAGATTACAGG (SEQ ID NO: 19), and preparedbased on the information of a gene data bank (E. coli Gene Bank), and anamplified fragment of about 2.2 kb including the guaC structural generegion covering SD-ATG and the translation termination codon was clonedbetween the HindIII site and the PstI site of pUC18 vector (TakaraShuzo). A HindIII site and a PstI site are respectively provided in thePCR primers.

The cloned guaC fragment of 2.2 kb contained one BglII site at about 1.1kb from the 5′ end, and therefore the plasmid was digested with BglII,blunt-ended by T4 DNA polymerase and ligated with T4 DNA ligase.Competent cells of E. coli JM109 were transformed with this ligationsolution, and transformants grown on LB agar plates containing 25 μg/mlof ampicillin were obtained. Plasmid DNAs were prepared from thetransformants of 18 clones, and a plasmid DNA from which a fragment ofabout 2.2 kp was excised by HindIII and PstI digestion, and whichfragment was not digested with BglII (pUC18guaC′ #1) was selected fromthe plasmid DNAs. The guaC contained in this plasmid has a frame shiftat the BglII site, and therefore it is predicted that the encoded enzymelacks its function (FIG. 3).

Then, the pUC18guaC′ #1 was digested with HindIII and PstI to prepare afragment of about 2.2 kb that included guaC. The fragment was insertedbetween the HindIII and PstI sites of pMAN997, which is a vector forhomologous recombination having a temperature-sensitive replicationorigin (tsori) (describe above), to obtain plasmid pMAN997guaC′ #1. Thestrain FADRadd-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻) and the strainFADRaddgsk (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, gsk⁻) were transformed at30° C. with the plasmid pMAN997guaC′ #1, and some of the obtainedcolonies were streaked on LB agar plates containing 25 μg/ml ofampicillin, and cultured at 30° C. overnight. Then, the culturedbacterial cells were plated on LB agar plates containing 25 μg/ml ofampicillin so that single colonies should be obtained, to obtaincolonies grown at 42° C. The procedure for obtaining single coloniesgrown at 42° C. was repeated once, and clones in which the whole plasmidwas integrated into the chromosome through homologous recombination wereselected. It was confirmed that these clones did not have the plasmid intheir cytoplasm. Then, several clones among these clones were streakedon LB agar plates, cultured at 30° C. overnight, then inoculated into LBliquid medium (3 ml/test tube), and cultured at 42° C. for 3 to 4 hourswith shaking. The culture was appropriately diluted so that singlecolonies should be obtained (about 10⁻⁵ to 10⁻⁶ dilution), plated on LBagar plates, and cultured at 42° C. overnight to obtain colonies. Onehundred colonies were randomly picked up from the emerged colonies, andeach allowed to grow on LB agar plates and LB agar plates containing 25μg/ml of ampicillin, respectively, and ampicillin-sensitive clones grownonly on the LB agar plates were selected. The fragments of about 2.2 kbincluding guaC were amplified by PCR from the chromosome DNA of thesetarget clones, and it was confirmed that the fragment was not digestedwith BglII. The clones satisfying the above conditions were consideredstrains deficient in guaC, and the clones derived from the strainsFADRadd-8-3 and FADRaddgsk are designated as FADRaddguaC (purF⁻, purA⁻,deoD⁻, purR⁻, add⁻, guaC⁻) and FADRaddgskguaC (purF⁻, purA⁻, deoD⁻,purR⁻, add⁻, gsk⁻, guaC⁻), respectively. It was also confirmed that theGMP reductase activity was not detected in them. The GMP reductaseactivity was measured according to the method of B. B. Garber et al. (J.Bacteriol., 43, 105 (1980)).

3) Evaluation of Purine Nucleoside-Producing Ability of DesensitizedType purF Plasmid-Introduced Strain

Transformants were produced by introducing pKFpurFKQ into the strainFADRaddguaC (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, guaC⁻) and FADRaddgskguaC(purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, gsk⁻, guaC⁻) produced in Section 2),and purine nucleoside-producing abilities of the strains were evaluated.The basal medium and the culture method for the purine nucleosideproduction and the analysis method were the same as Example 1. The MSmedium was supplemented with 5 mg/L adenine.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 8. A certain level of improvement in guanosineproduction was observed by the deficiency of guaC. TABLE 8 Evaluation ofpurine nucleoside-producing ability Purine nucleoside accumulationInosine Guanosine Host Plasmid (mg/L) (mg/L) FADRadd-8-3 pKFpurFKQ 10800 FADRaddguaC pKFpurFKQ 670 20 FADRaddgsk pKFpurFKQ 920 140FADRaddgskguaC pKFpurFKQ 750 180

Example 7 1) Breeding of Strain Deficient in 6-PhosphogluconateDehydrase Gene (edd)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, Perkin Elmer) by using thechromosome DNA of the strain W3110 and 29-mer primers for both ends,having nucleotide sequences of CTCGAATTCGGATATCTGGAAGAAGAGGG (SEQ ID NO:20) and CTCAAGCTTGGAATAGTCCCTTCGGTAGC (SEQ ID NO: 21), and preparedbased on the information obtained through searching of a gene data bank(E. coli Gene Bank) using “edd” as a key word, and an amplified fragmentof about 3.0 kb including the edd structural gene region covering ATGand the translation termination codon, about 810 bp 5′ upstream regionof ATG and about 360 bp downstream region of the translation terminationcodon was cloned into pCRTMII vector (Invitrogen) as it was. Theamplified fragment of the PCR product can be cloned into this vector asit is. The vector has EcoRI sites as restriction sites at vicinities ofthe both sides of the cloning site. A BamHI site and a HindIII site arerespectively provided in the PCR primers. The cloned edd fragment of 3.0kb contained two StuI sites at about 660 bp and 1900 bp from the 5′ end,and therefore the plasmid was digested with StuI. Then the StuI fragmentof about 1.25 kb was removed and DNA of the vector was subjected toself-ligation by T4 DNA ligase. Competent cells of E. coli HB101 weretransformed with this ligation solution, and transformants grown on LBagar plates containing 25 μg/ml of ampicillin were obtained. PlasmidDNAs were prepared from the transformants of 10 clones, and a plasmidDNA from which a fragment of about 1.25 kp was not excised by StuI(pCRTMIIedd′ #1) was selected from the plasmid DNAs. The edd containedin this plasmid has a deletion of a protein-coding region including apromoter region, and therefore it is predicted that the enzyme is notformed (FIG. 3).

Then, the pCRTMIIedd′ #1 was digested with EcoRI to prepare a fragmentof about 1.75 kb that included a part of edd and a flanking regionthereof. The fragment was inserted into the EcoRI site of pMAN997, whichis a vector for homologous recombination having a temperature-sensitivereplication origin (tsori) (describe above), to obtain plasmidpMAN997edd′ #1. The strain FADRadd-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻,add⁻) was transformed at 30° C. with the plasmid pMAN997edd′ #1, andsome of the obtained colonies were streaked on LB agar plates containing25 μg/ml of ampicillin, and cultured at 30° C. overnight. Then, thecultured bacterial cells were plated on LB agar plates containing 25μg/ml of ampicillin so that single colonies should be obtained, toobtain colonies grown at 42° C. The procedure for obtaining singlecolonies grown at 42° C. was repeated once, and clones in which thewhole plasmid was integrated into the chromosome through homologousrecombination were selected. It was confirmed that these clones did nothave the plasmid in their cytoplasm. Then, several clones among theseclones were streaked on LB agar plates, cultured at 30° C. overnight,then inoculated into LB liquid medium (3 ml/test tube), and cultured at42° C. for 3 to 4 hours with shaking. The culture was appropriatelydiluted so that single colonies should be obtained (about 10⁻⁵ to 10⁻⁶dilution), plated on LB agar plates, and cultured at 42° C. overnight toobtain colonies. One hundred colonies were randomly picked up from theemerged colonies, and each allowed to grow on LB agar plates and LB agarplates containing 25 μg/ml of ampicillin, respectively, andampicillin-sensitive clones grown only on the LB agar plates wereselected. The edd regions were amplified by PCR using the above PCRprimers from the chromosome DNA of these target clones, and the clonesin which the size of the amplified fragment is about 1.75 kb of deletiontype, not about 3.0 kb of wild type were selected. The clones wereconsidered strains deficient in edd and designated as FADRaddedd (purF⁻,purA⁻, deoD⁻, purR⁻, add⁻, edd⁻).

2) Evaluation of Purine Nucleoside-Producing Ability of DesensitizedType purF Plasmid-Introduced Strain

A transformant was produced by introducing pKFpurFKQ into the strainFADRaddedd (purF⁻, purA⁻, deoD⁻, purR⁻, add, edd⁻) bred in Section 1),and purine nucleoside-producing ability of the strain was evaluated. Thebasal medium and the culture method for the purine nucleoside productionand the analysis method were the same as Example 1. The MS medium wassupplemented with 5 mg/L adenine. The 6-phosphogulconate dehydraseencoded by edd is an enzyme which is induced by gluconic acid andpositioned at the first step in the Entner-Doudoroff pathwaymetabolizing gluconate to pyruvate. Because the gluconate was consideredto flow only into the pentose phosphate pathway by deficiency of thisenzyme, gluconic acid (48 g/L added) was used as a carbon source otherthan glucose to carry out the evaluation.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 9. Remarkable improvement in inosine production wasobserved by the deficiency of edd, when the gluconic acid was used asthe carbon source. The effect was also observed when glucose was used asthe carbon source. TABLE 9 Evaluation of purine nucleoside-producingability Purine nucleoside accumulation Carbon Inosine Guanosine HostPlasmid source (mg/L) (mg/L) FADRadd-8-3 pKFpurFKQ Glucose 1080 0FADRaddedd pKFpurFKQ Glucose 1340 0 FADRadd-8-3 pKFpurFKQ Gluconic 10500 acid FADRaddedd pKFpurFKQ Gluconic 2600 0 acid

Example 8 1) Breeding of Strain Deficient in Phosphoglucose IsomeraseGene (pgi)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, PerkinElmer) by using thechromosome DNA of the strain W3110 and 29-mer primers for both ends,having nucleotide sequences of CTCGTCGACTCCATTTTCAGCCTTGGCAC (SEQ ID NO:22) and CTCGCATGCGTCGCATCAGGCATCGGTTG (SEQ ID NO: 23), and preparedbased on the information obtained through searching of a gene data bank(E. coli Gene Bank) using “pgi” as a key word, and an amplified fragmentof about 2.2 kb including the pgi structural gene region covering ATGand the translation termination codon was cloned between the SalI siteand the SphI site of pUC18 vector (Takara Shuzo). A SalI site and a SphIsite are respectively provided in the PCR primers. The cloned pgifragment of 2.2 kb contained one BssHII site and one MluI site at about1170 bp and 1660 bp from the 5 end, respectively, and therefore theplasmid was digested with BssHII and MluI, and blunt-ended by T4 DNApolymerase. Then the fragment of about 500 bp between the BssHII siteand the MluI site was removed and DNA of the vector was subjected toself-ligation by T4DNA ligase. Competent cells of E. coli JM109weretransformed with this ligation solution, and transformants grown on LBagar plates containing 25 μg/ml of ampicillin were obtained. PlasmidDNAs were prepared from the transformants of 10 clones, and a plasmidDNA from which a fragment of about 1.7 kp was excised by SalI and SphIdigestion (pUC18 pgi′ #1) was selected from the plasmid DNAs. The pgicontained in this plasmid has a deletion between the BssHII site and theMluI site, and therefore it is predicted that the encoded enzyme lacksits function (FIG. 3).

Then, the pUC18 pgi′ #1 was digested with SalI and SphI to prepare afragment of about 1.7 kb that included pgi. The fragment was insertedbetween the SalI site and the SphI site of pMAN997, which is a vectorfor homologous recombination having a temperature-sensitive replicationorigin (tsori) (describe above), to obtain plasmid pMAN997 pgi′ #1. Thestrain FADRadd-8-3 (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻) and the strainFADRaddedd (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, edd⁻) were transformed at30° C. with the plasmid pMAN997 pgi′ #1, and some of the obtainedcolonies were each streaked on LB agar plates containing 25 μg/ml ofampicillin, and cultured at 30° C. overnight. Then, the culturedbacterial cells were plated on LB agar plates containing 25 μg/ml ofampicillin so that single colonies should be obtained, to obtaincolonies grown at 42° C. The procedure for obtaining single coloniesgrown at 42° C. was repeated once, and clones in which the whole plasmidwas integrated into the chromosome through homologous recombination wereselected. It was confirmed that these clones did not have the plasmid intheir cytoplasm. Then, several clones among these clones were streakedon LB agar plates, cultured at 30° C. overnight, then inoculated into LBliquid medium (3 ml/test tube), and cultured at 42° C. for 3 to 4 hourswith shaking. The culture was appropriately diluted so that singlecolonies should be obtained (about 10⁻⁵ to 10⁻⁶ dilution), plated on LBagar plates, and cultured at 42° C. overnight to obtain colonies. Onehundred colonies were randomly picked up from the emerged colonies, andeach allowed to grow on LB agar plates and LB agar plates containing 25μg/ml of ampicillin, respectively, and ampicillin-sensitive clones grownonly on the LB agar plates were selected. The pgi regions were amplifiedby PCR using the above PCR primers from the chromosome DNA of thesetarget clones, and the clones in which the size of the amplifiedfragment was about 1.7 kb of deletion type, not about 2.2 kb of wildtype were selected. The clones were considered strains deficient in pgi,and clones derived from FADRadd-8-3 and FADRaddedd were designated asFADRaddpgi (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, pgi⁻) and FADRaddeddpgi(purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, edd⁻, pgi⁻), respectively.

2) Evaluation of Purine Nucleoside-Producing Ability of DesensitizedType purF Plasmid-Introduced Strain

Transformants were produced by introducing pKFpurFKQ into the strainFADRaddpgi (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, pgi⁻) and the strainFADRaddeddpgi (purF⁻, purA⁻, deoD⁻, purR⁻, add, edd, pgi⁻) bred inSection 1), and purine nucleoside-producing abilities of the strainswere evaluated. The basal medium and the culture method for the purinenucleoside production and the analysis method were the same as Example 1provided that the amount of yeast extract in the MS medium (basalmedium) which was a medium used for evaluation of production wasincreased to 0.8%.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 10. By deficiency of pgi, growth remarkably loweredin the MS medium supplemented with 5 mg/L of adenine which was used inthe above Examples. Therefore, the medium in which the amount of yeastextract was increased to 0.8% was used. In this medium, the pgi⁺ parentstrain showed increase of the growth rate, lowering of inosineproduction and by-production of hypoxanthine. On the contrary,remarkable improvement in inosine production was observed in the straindeficient in pgi. TABLE 10 Evaluation of purine nucleoside-producingability Purine nucleoside accumulation Inosine Hypoxanthine Host Plasmid(mg/L) (mg/L) FADRadd-8-3 pKFpurFKQ 450 260 FADRaddpgi pKFpurFKQ 2770100 FADRaddedd pKFpurFKQ 780 210 FADRaddeddpgi pKFpurFKQ 3080 120

Example 9 1) Breeding of Strain Deficient in Adenine Deaminase Gene(yicP)

In a gene data bank (E. coli Gene Bank), yicP is registered as ORF (openreading frame, structural gene) which has a high homology with adeninedeaminase from Bacillus subtilis. PCR was carried out (94° C., 30 sec;55° C., 1 min; 72° C., 2 min; 30 cycles; Gene Amp PCR System Model 9600,Perkin Elmer) by using the chromosome DNA of the strain W3110 and 29-merprimers for both ends, having nucleotide sequences ofCTCCTGCAGCGACGTTTTCTTTTATGACA (SEQ ID NO: 24) andCTCAAGCTTCGTAACTGGTGACTTTTGCC (SEQ ID NO: 25), and prepared based on theinformation obtained through searching using “yicP” as a key word, toamplify a fragment of about 1.9 kb including the yicP structural generegion covering ATG and the translation termination codon, about 50 bp5′ upstream region of ATG and about 40 bp downstream region of thetranslation termination codon. A PstI site and a HindIII site arerespectively provided in the PCR primers. The PCR product was digestedwith PstI and HindIII, and cloned between the PstI site and the HindIIIsite of pUC18 vector (Takara Shuzo). The cloned yicP fragment of 1.9 kbcontained one HapI site and one EcoRV site at about 540 bp and 590 bpfrom the 5 end, respectively, and therefore the plasmid was digestedwith HapI and EcoRV. Then the HapI-EcoRV fragment of 47 bp was removedand DNA of the vector was subjected to self-ligation by T4 DNA ligase.Competent cells of E. coli JM109 were transformed with this ligationsolution, and transformants grown on LB agar plates containing 25 μg/mlof ampicillin were obtained. Plasmid DNAs were prepared from thetransformants of 10 clones, and a plasmid DNA which was not digestedwith HapI or EcoRV (pUC18yicP′ #1) was selected from the plasmid DNAs.The yicP contained in this plasmid has a frame shift due to a deletionof 47 bp of HapI-EcoRV sites, and therefore it is predicted that theencoded enzyme lacks its function (FIG. 3).

Then, the pUC18yicP′ #1 was digested with PstI and HindIII to prepare afragment of about 1.9 kb that included the yicP gene. The fragment wasinserted between the PstI site and the HindIII site of pMAN997, which isa vector for homologous recombination having a temperature-sensitivereplication origin (tsori) (describe above), to obtain plasmidpMAN997yicP′ #1. The strain FADRaddedd (purF⁻, purA⁻, deoD⁻, purR⁻,adds, edd⁻) was transformed at 30° C. with the plasmid pMAN997yicP′ #1,and some of the obtained colonies were streaked on LB agar platescontaining 25 μg/ml of ampicillin, and cultured at 30° C. overnight.Then, the cultured bacterial cells were plated on LB agar platescontaining 25 μg/ml of ampicillin so that single colonies should beobtained, to obtain colonies grown at 42° C. The procedure for obtainingsingle colonies grown at 42° C. was repeated once, and clones in whichthe whole plasmid was integrated into the chromosome through homologousrecombination were selected. It was confirmed that these clones did nothave the plasmid in their cytoplasm. Then, several clones among theseclones were streaked on LB agar plates, cultured at 30° C. overnight,then inoculated into LB liquid medium (3 ml/test tube), and cultured at42° C. for 3 to 4 hours with shaking. The culture was appropriatelydiluted so that single colonies should be obtained (about 10⁻⁵ to 10⁻⁶dilution), plated on LB agar plates, and cultured at 42° C. overnight toobtain colonies. One hundred colonies were randomly picked up from theemerged colonies, and each allowed to grow on LB agar plates and LB agarplates containing 25 μg/ml of ampicillin, respectively, andampicillin-sensitive clones grown only on the LB agar plates wereselected. The yicP regions were amplified by PCR using the above PCRprimers from the chromosome DNA of these target clones, and the clonesin which the size of the amplified fragment was not digested with HapIor EcoRV were selected. It was also confirmed that the adenine deaminaseactivity was not detected in these clones. The adenine deaminaseactivity was measured according to the method of Per Nygaard et al. (J.Bacteriol., 178, 846-853 (1996)) The clones were considered strainsdeficient in yicP, and designated as FADRaddeddyicP (purF⁻, purA⁻,deoD⁻, purR⁻, add⁻, edd⁻, yicP⁻).

2) Breeding of Strain Deficient in Phosphoglucose Isomerase Gene (pgi)from Strain Deficient in Adenine Deaminase Gene (yicP)

The deficiency of pgi was also added to the strain FADRaddeddyicP(purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, edd⁻, yicP⁻). By using pMAN997 pgi′#1 constructed in Example 8, a strain FADRaddeddyicPpgi (purF⁻, purA⁻,deoD⁻, purR⁻, add⁻, edd⁻, yicP⁻, pgi⁻) was obtained in the same methodas in Example 8.

3) Evaluation of Purine Nucleoside-Producing Ability of DesensitizedType purF Plasmid-Introduced Strain

Transformants were produced by introducing pKFpurFKQ into the strainFADRaddeddyicP (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, edd⁻, yicP⁻) and thestrain FADRaddeddyicPpgi (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, edd⁻, yicP⁻,pgi⁻) bred in Sections 1) and 2), and responses of growth to an adenineamount and purine nucleoside-producing abilities of the strains wereevaluated. The basal medium and the culture method for the purinenucleoside production and the analysis method were the same as Example 1provided that the used medium was the MS medium to which adenine wasadded in an amount between 0 to 50 mg/L.

The results of the evaluation of the growth response to adenine and thepurine nucleoside-producing ability are shown in Table 11. By deficiencyof yicP, the growth rate with respect to adenine was improved and aneffect of the deficiency of yicP was observed when adenine was added inamounts of 50 mg/L and 20 mg/L. TABLE 11 Evaluation of purinenucleoside-producing ability Purine nucleoside Adenine Growthaccumulation added rate Inosine Hypoxanthine Host Plasmid (mg/L) (OD)(mg/L) (mg/L) 0 2.2 870 0 FADRaddedd pKFpurFKQ 50 3.2 650 0 0 2.4 870 0FADRaddeddyicP pKFpurFKQ 50 6.8 1100 40 5 2.2 1420 28 FADRaddeddpgipKFpurFKQ 20 3.4 1760 48 5 2.1 1380 7 FADRaddeddyicPpgi pKFpurFKQ 20 3.72350 19

Example 10 1) Preparation of PRPP Synthetase Gene (prs)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, PerkinElmer) by using thechromosome DNA of the strain W3110 and 38-mer and 29-mer primers forboth ends, having nucleotide sequences ofCTCGTCGACTGCCTAAGGATCTTCTCATGCCTGATATG (SEQ ID NO: 26) andCTCGCATGCGCCGGGTTCGATTAGTGTTC (SEQ ID NO: 27), and prepared based on theinformation of a gene data bank (E. coli Gene Bank), and an amplifiedfragment of about 1 kb including the prs structural gene region coveringSD-ATG and the translation termination codon was cloned into pUC18vector (Takara Shuzo). A SalI site and a SphI site are respectivelyprovided in the PCR primers. The PCR product was digested with SalI andSphI, and cloned between the SalI site and the SphI site of pUC18 vector(pUCprs).

2) Construction of Desensitized Type prs

A prs fragment was excised from the plasmid carrying the prs of about 1kb cloned in Section 1) by SalI and SphI digestion, and inserted betweenthe SalI and SphI sites of the multi-cloning site of a plasmid forintroducing mutation, pKF19k (Takara Shuzo) to obtain the target clone(pKFprs). S. G. Bower et al. (J. Biol. Chem., 264, 10287 (1989)) hassuggested that PRPP synthetase (Prs) is subjected to feedback inhibitionby AMP and ADP. It is also described that the enzyme whose Asp (D) atposition 128 is mutated to Ala (A) is partially desensitized. Therefore,the following synthetic DNA primer was prepared for gene substitutionrealizing mutation of Asp (D) at position 128 PRPP synthetase (Prs) toAla (A), and pKFprs was subjected to site-directed mutagenesis accordingto the protocol of Site-directed Mutagenesis System Mutan-Super ExpressKm (Takara Shuzo) to introduce a site-directed mutation into the pKFprs.Primer for D128A mutation: 5′-GCGTGCAGAGCCACTATCAGC-3′ (SEQ ID NO: 28)

After the mutagenesis, 12 clones were randomly picked up from theresulting transformants, and plasmids were produced from them. Bynucleotide sequencing of the plasmids around the locations where themutations were introduced, it was confirmed that 9 clones of targetmutants were obtained. The prs fragment was excised with SalI and SphIfrom pKFprsDA having the mutant type prs, and inserted between the SalIsite and the SphI site of pUC18 and pSTV18 (Takara Shuzo). For using thewild type prs as a control, the prs fragment was excised with SalI andSphI from pUCprs constructed in the above, and inserted between the SalIsite and the SphI site of pSTV18 (Takara Shuzo). Each of the plasmidspUCprsDA and pSTVprsDA and the plasmids puCprs and pSTVprs has aninserted mutant prs or wild type prs downstream of the lacp/o (promoterof lactose operon) derived from pUC18 and pSTV18, respectively, and theprs is expressed under the control of this promoter.

Recombinant bacteria obtained by transforming E. coli JM109 cells withthe above four plasmids were cultured in LB liquid medium for eighthours, and collected, and crude enzyme extracts were prepared from them.The PRPP synthetase activity of the extracts and degrees of inhibitionby ADP were measured according to the method of K. F. Jensen et al.(Analytical Biochemistry, 98, 254-263 (1979)) which was partiallymodified. Specifically, [α-³²P]ATP was used as the substrate and[³²P]AMP produced by the reaction was measured. The results are shown inTable 12. TABLE 12 PRPP synthetase (Prs) activity Specific activity(nmole/min/mg crude enzyme extract) Host Plasmid Property None 5 mM ADPJM109 pUC18 Control 2.9 ND JM109 pUCprs High copy, 75.9 ND wild typeJM109 pUCprsDA High copy, 80.8 20.2 mutant type JM109 pSTVprs Mediumcopy, 11.5 ND wild type JM109 pSTVprsDA Medium copy, 10.6 2.7  mutanttype

3) Evaluation of Purine Nucleoside-Producing Ability of DesensitizedType prs Plasmid-Introduced Strain

Strains each having two plasmids simultaneously were made by introducingpKFpurFKQ into the strain FADRaddeddyicPpgi (purF⁻, purA⁻, deoD⁻, purR⁻,add⁻, eddy, yicP⁻, pgi⁻) bred in Example 9, Section 3) to obtain atransformant and further each introducing pSTVprs and pSTVprsDA carryingprs and prsDA genes into the transformant, and purinenucleoside-producing abilities of the strains were evaluated. The basalmedium and the culture method for the purine nucleoside production andthe analysis method were the same as Example 1 provided that the amountof yeast extract in the MS medium was increased to 0.4%.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 13. By introduction of mutant prsDA as a plasmid, aneffect of improvement in inosine production was observed. TABLE 13Evaluation of purine nucleoside-producing ability Purine nucleosideaccumulation Inosine Hypoxanthine Host Plasmid (mg/L) (mg/L)FADRaddeddyicPpgi pKFpurFKQ 1600 8 pKFpurFKQ + 1450 3 pSTVprspKFpurFKQ + 1815 10 pSTVprsDA

Example 11 1) Breeding of Strain Deficient in Xanthosine PhosphorylaseGene (xapA)

A gene inactivated by mutation was constructed in one step by Cross-overPCR using four primers prepared based on the information obtainedthrough searching of a gene data bank (E. Coli Gene Bank) using “xapA”as a key word. The used primers are as follows: N-out: (SEQ ID NO: 29)5′-CGCGGATCCGCGACATAGCCGTTGTCGCC-3′ N-in: (SEQ ID NO: 30)5′-CCCATCCACTAAACTTAAACATCGTGGCGTGAAATCAGG-3′ C-in: (SEQ ID NO: 31)5′-TGTTTAAGTTTAGTGGATGGGCATCAACCTTATTTGTGG-3′ C-out: (SEQ ID NO: 32)5′-CGCAAGCTTCAAACTCCGGGTTACGGGCG-3′

First, PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2min; 30 cycles; Gene Amp PCR System Model 9600, Perkin Elmer) by usingthe chromosome DNA of the strain W3110 and primers N-out (29-mer) andN-in (39-mer) as well as primers C-in (39-mer) and C-out (29-mer) forboth ends, to obtain two PCR products (both are fragments of about 850bp), respectively. Then, PCR was again carried out by using a mixture ofthe two PCR products as a template and the primers N-out and C-out forboth ends, to amplify a gene fragment in which the gene region includingthe xapA structural gene region was shortened from a fragment of about2.4 kb (size of wild type) to a fragment of about 1.7 kb. A BamHI siteand a HindIII site are provided in the PCR primers N-out and C-out,respectively. This PCR product was digested with BamHI and HindIII, andthe obtained fragment was ligated by T4 DNA ligase with a plasmidobtained by digesting pMAN997, which is a vector for homologousrecombination having a temperature-sensitive replication origin (tsori)(describe above), with BamHI and HindIII. Competent cells of E. coliJM109 were transformed with this ligation solution, and transformantsgrown on LB agar plates containing 25 μg/ml of ampicillin were obtained.Plasmid DNAs were prepared from the transformants of 10 clones, and aplasmid DNA from which a fragment of about 1.7 kp was excised by BamHIand HindIII digestion (pMAN997xapA′ #1) was selected from the plasmidDNAs. The xapA contained in this plasmid has a deletion of about 700 bpin the structural gene, and therefore it is predicted that the encodedenzyme lacks its function (FIG. 3).

The strain FADRaddeddyicPpgi (purF⁻, purA⁻, deoD⁻, purR⁻, adds, eddy,yicP⁻, pgi⁻) was transformed at 30° C. with the plasmid pMAN997xapA′ #1,and some of the obtained colonies were streaked on LB agar platescontaining 25 μg/ml of ampicillin, and cultured at 30° C. overnight.Then, the cultured bacterial cells were plated on LB agar platescontaining 25 μg/ml of ampicillin so that single colonies should beobtained, to obtain colonies grown at 42° C. The procedure for obtainingsingle colonies grown at 42° C. was repeated once, and clones in whichthe whole plasmid was integrated into the chromosome through homologousrecombination were selected. It was confirmed that these clones did nothave the plasmid in their cytoplasm. Then, several clones among theseclones were streaked on LB agar plates, cultured at 30° C. overnight,then inoculated into LB liquid medium (3 ml/test tube), and cultured at42° C. for 3 to 4 hours with shaking. The culture was appropriatelydiluted so that single colonies should be obtained (about 10⁵ to 10⁶dilution), plated on LB agar plates, and cultured at 42° C. overnight toobtain colonies. One hundred colonies were randomly picked up from theemerged colonies, and each allowed to grow on LB agar plates and LB agarplates containing 25 μg/ml of ampicillin, respectively, andampicillin-sensitive clones grown only on the LB agar plates wereselected. The xapA region were amplified by PCR using the above PCRprimers N-out and C-out from the chromosome DNA of these target clones,and the clones in which the size of the amplified fragment was about 1.7kb were selected. The clones were considered strains deficient in xapA,and designated as FADRaddeddyicPpgixapA (purF⁻, purA⁻, deoD⁻, purR⁻,add⁻, edd⁻, yicP⁻, pgi⁻, xapA⁻). In the strain deficient in xapA,xanthine production in a medium was not observed by culture withxanthosine supplemented, and it was confirmed that xanthosinephosphorylase was not induced. The xanthosine phosphorylase activity wasmeasured according to the method of K. Hammer Jespersen et al. (Molec.Gen. Genet., 179, 341-348 (1980)).

2) Evaluation of Purine Nucleoside-Producing Ability of DesensitizedType purF Plasmid-Introduced Strain

A transformant was produced by introducing pKFpurFKQ into the strainFADRaddeddyicPpgixapA (purF⁻, purA⁻, deoD⁻, purR⁻, add, edd⁻, yicP⁻,pgi⁻, xapA⁻) bred in Section 1), and purine nucleoside-producing abilityof the strain was evaluated. The basal medium and the culture method forthe purine nucleoside production and the analysis method were the sameas Example 1 provided that the MS medium in which the amount of yeastextract was increased to 0.8% was used.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 14. When the amount of yeast extract in the MS mediumwas increased, by-production of hypoxanthine which remarkably occurredafter sugar consumption in the latter half of the culture was reduced bydeficiency of xapA, and improvement of inosine production was observed.TABLE 14 Evaluation of purine nucleoside-producing ability Purinenucleoside Culture accumulation period Inosine Hypoxanthine Host Plasmid(days) (mg/L) (mg/L) FADRaddeddyicPpgi pKFpurFKQ 3 4640 146 6 1850 1500FADRaddeddyicPpgixapA pKFpurFKQ 3 5870 57 6 3810 915

Example 12 1) Breeding of Strain Deficient in Nucleoside Permease Gene(nupG)

PCR was carried out (94° C., 30 sec; 55° C., 1 min; 72° C., 2 min; 30cycles; Gene Amp PCR System Model 9600, Perkin Elmer) by using thechromosome DNA of the strain W3110 as a template and 35-mer primers forboth ends, having nucleotide sequences ofCTCGAATTCATGGTGCCGAACCACCTTGATAAACG (SEQ ID NO: 33) andCTCGTCGACATGCCGAAACCGGCGAATATAGCGAC (SEQ ID NO: 34), and prepared basedon the information of a gene data bank (E. coli Gene Bank), to amplify afragment of about 2.7 kb of an nupG structural gene region coveringSD-ATG and the translation termination codon. An EcoRI site and a SalIsite are respectively provided in the PCR primers. The amplifiedfragment was digested with EcoRI, SalI and AflII. Since thePCR-amplified fragment contained two AflII sites, three fragments ofabout 750 bp, 820 bp and 1130 bp were formed. The two fragments of about720 bp and 1130 bp other than the AflII fragment of about 820 bp werecollected and were ligated by T4 DNA ligase with DNA obtained bydigesting pUC18 vector (Takara Shuzo) with EcoRI and SalI. Competentcells of E. coli HB101 were transformed with this ligation solution, andplasmid DNAs were prepared from 16 of the emerged colonies, and aplasmid DNA in which a fragment digested with EcoRI and SalI was ofabout 1.9 kb (pUC18nupG′ #1) was selected from the plasmid DNAs. ThepUC18nupG′ #1 was digested with EcoRI and SalI, and the resultingfragment of about 1.9 kb was ligated by T4 DNA ligase with a plasmidobtained by digesting pMAN997, which is a vector for homologousrecombination having a temperature-sensitive replication origin (tsori)(described above), with EcoRI and SalI. Competent cells of E. coli JM109were transformed with this ligation solution, and transformants grown onLB agar plates containing 25 μg/ml of ampicillin were obtained. PlasmidDNAs were prepared from the transformants of 10 clones, and a plasmidDNA from which a fragment of about 1.9 kp was excised by EcoRI and SalIdigestion (pMAN997nupG′ #1) was selected from the plasmid DNAs. The nupGcontained in this plasmid DNA has a deletion of about 820 bp in thestructural gene, and therefore it is predicted that the encoded enzymelacks its function (FIG. 3).

The strain FADRaddeddyicPpgi (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, edd⁻,yicP⁻, pgi⁻) was transformed at 30° C. with the plasmid pMAN997nupG′ #1,and some of the obtained colonies were streaked on LB agar platescontaining 25 μg/ml of ampicillin, and cultured at 30° C. overnight.Then, the cultured bacterial cells were plated on LB agar platescontaining 25 μg/ml of ampicillin so that single colonies should beobtained, to obtain colonies grown at 42° C. The procedure for obtainingsingle colonies grown at 42° C. was repeated once, and clones in whichthe whole plasmid was integrated into the chromosome through homologousrecombination were selected. It was confirmed that these clones did nothave the plasmid in their cytoplasm. Then, several clones among theseclones were streaked on LB agar plates, cultured at 30° C. overnight,then inoculated into LB liquid medium (3 ml/test tube), and cultured at42° C. for 3 to 4 hours with shaking. The culture was appropriatelydiluted so that single colonies should be obtained (about 10⁻⁵ to 10⁻⁶dilution), plated on LB agar plates, and cultured at 42° C. overnight toobtain colonies. One hundred colonies were randomly picked up from theemerged colonies, and each allowed to grow on LB agar plates and LB agarplates containing 25 μg/ml of ampicillin, respectively, andampicillin-sensitive clones grown only on the LB agar plates wereselected. The nupG region were amplified by PCR using the above PCRprimers from the chromosome DNA of these target clones, and clones inwhich the size of the amplified fragment was about 1.9 kb were selected.These clones were considered strains deficient in nupG, and designatedas FADRaddeddyicPpginupG (purF⁻, purA⁻, deoD⁻, purR⁻, add⁻, eddy, yicP⁻,pgi⁻, nupG⁻).

2) Evaluation of Purine Nucleoside-Producing Ability of DesensitizedType purF-Introduced Strain

A transformant was made by introducing pKFpurFKQ into the strainFADRaddeddyicPpginupG (purF⁻, purA⁻, deoD⁻, purR⁻, add, edd, yicP⁻,pgi⁻, nupG⁻) bred in Section 1), and purine nucleoside-producing abilityof the strain was evaluated. The basal medium and the culture method forthe purine nucleoside production and the analysis method were the sameas Example 1 provided that the MS medium in which the amount of yeastextract was increased to 1.2%.

The results of the evaluation of the purine nucleoside-producing abilityare shown in Table 15. When the amount of yeast extract in the MS mediumwas increased, by-production of hypoxanthine which remarkably occurredafter sugar consumption in the latter half of the culture was reducedand improvement of inosine production was observed by deficiency ofnupG. TABLE 15 Evaluation of purine nucleoside-producing ability Purinenucleoside accumulation Inosine Hypoxanthine Host Plasmid (mg/L) (mg/L)FADRaddeddyicPpgi pKFpurFKQ 1190 835 FADRaddeddyicPpginupG pKFpurFKQ3390 315

INDUSTRIAL APPLICABILITY

According to the present invention, a purine nucleoside-producingbacterium is created by derepressing and desensitizing an enzyme whichsubjected to the control in purine nucleoside biosynthesis and furtherblocking a decomposition system and a conversion system. The createdpurine nucleoside-producing bacterium can be suitably used forproduction of a purine nucleoside by fermentation.

1.-13. (canceled)
 14. A method for producing a purine nucleoside byfermentation comprising: culturing a microorganism belonging to thegenus Escherichia in a culture medium to produce and accumulate thepurine nucleoside in the medium, and collecting the purine nucleoside,wherein said microorganism has purine nucleoside-producing ability andis modified to block a reaction catalyzed by succinyl-adenosinemonophosphate synthase.
 15. The method according to claim 14, whereinsaid microorganism is further modified to increase an activity of anenzyme involved in purine nucleoside biosynthesis in cells of themicroorganism.
 16. The method according to claim 14, wherein saidmicroorganism is further modified to increase an expression amount of agene for an enzyme involved in purine nucleoside biosynthesis.
 17. Themethod according to claim 14, wherein said microorganism is furthermodified to deregulate control of an enzyme involved in the purinenucleoside biosynthesis.
 18. The method according to claim 14, whereinsaid microorganism is further modified to desensitize feedbackinhibition of an enzyme involved in the purine nucleoside biosynthesis.19. The method according to claim 18, wherein the enzyme involved in thepurine nucleoside biosynthesis is phosphoribosyl pyrophosphateamidotransferase.
 20. The method according to claim 16, wherein theenzyme involved in the purine nucleoside biosynthesis is phosphoribosylpyrophosphate amidotransferase or phosphoribosyl pyrophosphatesynthetase.
 21. The method according to claim 14, wherein saidmicroorganism is further modified to inactivate a purine repressor. 22.The method according to claim 14, wherein said microorganism is furthermodified to block a reaction catalyzed by an enzyme selected from thegroup consisting of purine nucleoside phosphorylase, adenosinedeaminase, inosine-guanosine kinase, guanosine monophosphate reductase,6-phosphogluconoate deydrase, phosphoglucose isomerase, adeninedeaminase, and xanthosine phosphorylase.
 23. The method according toclaim 14, wherein said microorganism is further modified to weakenincorporation of a purine nucleoside into cells of the microorganism.24. The method according to claim 23, wherein the incorporation of thepurine nucleoside into cells of the microorganism is weakened byblockage of a reaction involved in the incorporation of the purinenucleoside into cells of the microorganism, and the reaction involved inthe incorporation of the purine nucleoside into cells of themicroorganism is a reaction catalyzed by nucleoside permease.
 25. Themethod according to claim 14, wherein said purine nucleoside is a purinenucleoside selected from the group consisting of inosine and guanosine.